CRISPR enzymes and systems

ABSTRACT

The invention provides for systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides non-naturally occurring or engineered DNA or RNA-targeting systems comprising a novel DNA or RNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is a continuation-in-part of international patentapplication Serial No. PCT/US2016/038238 filed Jun. 17, 2016, whichpublished as PCT Publication No. WO2016/205749 on Dec. 22, 2016 andwhich claims the benefit of U.S. Provisional Patent Application Nos.62/181,663, filed Jun. 18, 2015 and 62/245,264, filed Oct. 22, 2015.

All documents cited or referenced in herein cited documents, togetherwith any manufacturer's instructions, descriptions, productspecifications, and product sheets for any products mentioned herein orin any document incorporated by reference herein, are herebyincorporated herein by reference, and may be employed in the practice ofthe invention. More specifically, all referenced documents areincorporated by reference to the same extent as if each individualdocument was specifically and individually indicated to be incorporatedby reference.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numbersMH100706, MH110049, DK097768, GM010407 awarded by the NationalInstitutes of Health. The government has certain rights in theinvention.

SEQUENCE LISTING

The instant application contains a sequence listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 9, 2016, isnamed 47627_99_2136_SL.txt and is 1,027,261 bytes in size.

FIELD OF THE INVENTION

The present invention generally relates to systems, methods andcompositions used for the control of gene expression involving sequencetargeting, such as perturbation of gene transcripts or nucleic acidediting, that may use vector systems related to Clustered RegularlyInterspaced Short Palindromic Repeats (CRISPR) and components thereof.

BACKGROUND OF THE INVENTION

Recent advances in genome sequencing techniques and analysis methodshave significantly accelerated the ability to catalog and map geneticfactors associated with a diverse range of biological functions anddiseases. Precise genome targeting technologies are needed to enablesystematic reverse engineering of causal genetic variations by allowingselective perturbation of individual genetic elements, as well as toadvance synthetic biology, biotechnological, and medical applications.Although genome-editing techniques such as designer zinc fingers,transcription activator-like effectors (TALEs), or homing meganucleasesare available for producing targeted genome perturbations, there remainsa need for new genome engineering technologies that employ novelstrategies and molecular mechanisms and are affordable, easy to set up,scalable, and amenable to targeting multiple positions within theeukaryotic genome. This would provide a major resource for newapplications in genome engineering and biotechnology.

The CRISPR-Cas systems of bacterial and archaeal adaptive immunity showextreme diversity of protein composition and genomic loci architecture.The CRISPR-Cas system loci has more than 50 gene families and there isno strictly universal genes indicating fast evolution and extremediversity of loci architecture. So far, adopting a multi-prongedapproach, there is comprehensive cas gene identification of about 395profiles for 93 Cas proteins. Classification includes signature geneprofiles plus signatures of locus architecture. A new classification ofCRISPR-Cas systems is proposed in which these systems are broadlydivided into two classes, Class 1 with multisubunit effector complexesand Class 2 with single-subunit effector modules exemplified by the Cas9protein (FIGS. 1A and 1B). Novel effector proteins associated with Class2 CRISPR-Cas systems may be developed as powerful genome engineeringtools and the prediction of putative novel effector proteins and theirengineering and optimization is important.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

There exists a pressing need for alternative and robust systems andtechniques for targeting nucleic acids or polynucleotides (e.g. DNA orRNA or any hybrid or derivative thereof) with a wide array ofapplications. This invention addresses this need and provides relatedadvantages. Adding the novel DNA or RNA-targeting systems of the presentapplication to the repertoire of genomic and epigenomic targetingtechnologies may transform the study and perturbation or editing ofspecific target sites through direct detection, analysis andmanipulation. To utilize the DNA or RNA-targeting systems of the presentapplication effectively for genomic or epigenomic targeting withoutdeleterious effects, it is critical to understand aspects of engineeringand optimization of these DNA or RNA targeting tools.

The invention provides a method of modifying sequences associated withor at a target locus of interest, the method comprising delivering tosaid locus a non-naturally occurring or engineered compositioncomprising a Type V CRISPR-Cas loci effector protein and one or morenucleic acid components, wherein the effector protein forms a complexwith the one or more nucleic acid components and upon binding of thesaid complex to the locus of interest the effector protein induces themodification of the sequences associated with or at the target locus ofinterest. In a preferred embodiment, the modification is theintroduction of a strand break. In a preferred embodiment, the sequencesassociated with or at the target locus of interest comprises DNA and theeffector protein is encoded by a subtype V-A CRISPR-Cas loci or asubtype V-B CRISPR-Cas loci or a subtype V-C CRISPR-Cas loci.

It will be appreciated that the terms Cas enzyme, CRISPR enzyme, CRISPRprotein Cas protein and CRISPR Cas are generally used interchangeablyand at all points of reference herein refer by analogy to novel CRISPReffector proteins further described in this application, unlessotherwise apparent, such as by specific reference to Cas9. The CRISPReffector proteins described herein are preferably C2c1 or C2c3 effectorproteins.

The invention provides a method of modifying sequences associated withor at a target locus of interest, the method comprising delivering tosaid sequences associated with or at the locus a non-naturally occurringor engineered composition comprising a C2c1 or C2c3 loci effectorprotein and one or more nucleic acid components, wherein the C2c1 orC2c3 effector protein forms a complex with the one or more nucleic acidcomponents and upon binding of the said complex to the locus of interestthe effector protein induces the modification of sequences associatedwith or at the target locus of interest. In a preferred embodiment, themodification is the introduction of a strand break. In a preferredembodiment the C2c1 or C2c3 effector protein forms a complex with onenucleic acid component; advantageously an engineered or non-naturallyoccurring nucleic acid component. The induction of modification ofsequences associated with or at the target locus of interest can be C2c1or C2c3 effector protein-nucleic acid guided. In a preferred embodimentthe one nucleic acid component is a CRISPR RNA (crRNA). In a preferredembodiment the one nucleic acid component is a mature crRNA or guideRNA, wherein the mature crRNA or guide RNA comprises a spacer sequence(or guide sequence) and a direct repeat sequence or derivatives thereof.In a preferred embodiment the spacer sequence or the derivative thereofcomprises a seed sequence, wherein the seed sequence is critical forrecognition and/or hybridization to the sequence at the target locus. Ina preferred embodiment, the sequences associated with or at the targetlocus of interest comprise linear or super coiled DNA.

Aspects of the invention relate to C2c1 or C2c3 effector proteincomplexes having one or more non-naturally occurring or engineered ormodified or optimized nucleic acid components. In a preferred embodimentthe nucleic acid component of the complex may comprise a guide sequencelinked to a direct repeat sequence, wherein the direct repeat sequencecomprises one or more stem loops or optimized secondary structures. Incertain embodiments, the direct repeat has a minimum length of 16 ntsand a single stem loop. In further embodiments the direct repeat has alength longer than 16 nts, preferably more than 17 nts, and has morethan one stem loop or optimized secondary structures. In a preferredembodiment the direct repeat may be modified to comprise one or moreprotein-binding RNA aptamers. In a preferred embodiment, one or moreaptamers may be included such as part of optimized secondary structure.Such aptamers may be capable of binding a bacteriophage coat protein.The bacteriophage coat protein may be selected from the group comprisingQβ, F2, GA, fr, JP501, MS2, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1,TW18, VK, SP, FL, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r,7s and PRR1. In a preferred embodiment the bacteriophage coat protein isMS2. The invention also provides for the nucleic acid component of thecomplex being 30 or more, 40 or more or 50 or more nucleotides inlength.

The invention provides methods of genome editing wherein the methodcomprises two or more rounds of C2c1 or C2c3 effector protein targetingand cleavage. In certain embodiments, a first round comprises the C2c1or C2c3 effector protein cleaving sequences associated with a targetlocus far away from the seed sequence and a second round comprises theC2c1 or C2c3 effector protein cleaving sequences at the target locus. Inpreferred embodiments of the invention, a first round of targeting by aC2c1 or C2c3 effector protein results in an indel and a second round oftargeting by the C2c1 or C2c3 effector protein may be repaired viahomology directed repair (HDR). In a most preferred embodiment of theinvention, one or more rounds of targeting by a C2c1 or C2c3 effectorprotein results in staggered cleavage that may be repaired withinsertion of a repair template.

The invention provides methods of genome editing or modifying sequencesassociated with or at a target locus of interest wherein the methodcomprises introducing a C2c1 or C2c3 effector protein complex into anydesired cell type, prokaryotic or eukaryotic cell, whereby the C2c1 orC2c3 effector protein complex effectively functions to integrate a DNAinsert into the genome of the eukaryotic or prokaryotic cell. Inpreferred embodiments, the cell is a eukaryotic cell and the genome is amammalian genome. In preferred embodiments the integration of the DNAinsert is facilitated by non-homologous end joining (NHEJ)-based geneinsertion mechanisms. In preferred embodiments, the DNA insert is anexogenously introduced DNA template or repair template. In one preferredembodiment, the exogenously introduced DNA template or repair templateis delivered with the C2c1 or C2c3 effector protein complex or onecomponent or a polynucleotide vector for expression of a component ofthe complex. In a more preferred embodiment the eukaryotic cell is anon-dividing cell (e.g. a non-dividing cell in which genome editing viaHDR is especially challenging). In preferred methods of genome editingin human cells, the C2c1 or C2c3 effector proteins may include but arenot limited to the specific species of C2c1 or C2c3 effector proteinsdisclosed herein.

The invention also provides a method of modifying a target locus ofinterest, the method comprising delivering to said locus a non-naturallyoccurring or engineered composition comprising a C2c1 loci effectorprotein and one or more nucleic acid components, wherein the C2c1effector protein forms a complex with the one or more nucleic acidcomponents and upon binding of the said complex to the locus of interestthe effector protein induces the modification of the target locus ofinterest. In a preferred embodiment, the modification is theintroduction of a strand break.

The invention also provides a method of modifying a target locus ofinterest, the method comprising delivering to said locus a non-naturallyoccurring or engineered composition comprising a C2c3 loci effectorprotein and one or more nucleic acid components, wherein the C2c3effector protein forms a complex with the one or more nucleic acidcomponents and upon binding of the said complex to the locus of interestthe effector protein induces the modification of the target locus ofinterest. In a preferred embodiment, the modification is theintroduction of a strand break.

In such methods the target locus of interest may be comprised within aDNA molecule. In such methods the target locus of interest may becomprised in a DNA molecule in vitro.

In such methods the target locus of interest may be comprised in a DNAmolecule within a cell. The cell may be a prokaryotic cell or aeukaryotic cell. The cell may be a mammalian cell. The mammalian cellmany be a non-human primate, bovine, porcine, rodent or mouse cell. Thecell may be a non-mammalian eukaryotic cell such as poultry, fish orshrimp. The cell may also be a plant cell. The plant cell may be of acrop plant such as cassava, corn, sorghum, wheat, or rice. The plantcell may also be of an algae, tree or vegetable. The modificationintroduced to the cell by the present invention may be such that thecell and progeny of the cell are altered for improved production ofbiologic products such as an antibody, starch, alcohol or other desiredcellular output. The modification introduced to the cell by the presentinvention may be such that the cell and progeny of the cell include analteration that changes the biologic product produced.

The mammalian cell many be a non-human mammal, e.g., primate, bovine,ovine, porcine, canine, rodent, Leporidae such as monkey, cow, sheep,pig, dog, rabbit, rat or mouse cell. The cell may be a non-mammalianeukaryotic cell such as poultry bird (e.g., chicken), vertebrate fish(e.g., salmon) or shellfish (e.g., oyster, claim, lobster, shrimp) cell.The cell may also be a plant cell. The plant cell may be of a monocot ordicot or of a crop or grain plant such as cassava, com, sorghum,soybean, wheat, oat or rice. The plant cell may also be of an algae,tree or production plant, fruit or vegetable (e.g., trees such as citrustrees, e.g., orange, grapefruit or lemon trees; peach or nectarinetrees; apple or pear trees; nut trees such as almond or walnut orpistachio trees; nightshade plants; plants of the genus Brassica; plantsof the genus Lactuca; plants of the genus Spinacia; plants of the genusCapsicum; cotton, tobacco, asparagus, carrot, cabbage, broccoli,cauliflower, tomato, eggplant, pepper, lettuce, spinach, strawberry,blueberry, raspberry, blackberry, grape, coffee, cocoa, etc).

The invention also provides an engineered CRISPR protein, complex,composition, system, vector, cell or cell line according to theinvention for use in modifying a locus of interest in a cell. Saidmodifying preferably comprises contacting the cell with any of theabove-described compositions or any of the above-described systems. Theinvention also provides a use of an engineered CRISPR protein, complex,composition, system, vector, cell or cell line according to theinvention in the preparation of a medicament for modifying a locus ofinterest in a cell.

The invention also provides an engineered CRISPR protein, complex,composition, system, vector, cell or cell line according to theinvention for use in modifying a locus of interest in a cell. Theinvention also provides a use of an engineered CRISPR protein, complex,composition, system, vector, cell or cell line according to theinvention in the preparation of a medicament for modifying a locus ofinterest in a cell. Said modifying preferably comprises contacting thecell with any of the above-described compositions or any of theabove-described systems.

The invention provides a method of modifying a target locus of interest,the method comprising delivering to said locus a non-naturally occurringor engineered composition comprising a Type VI CRISPR-Cas loci effectorprotein and one or more nucleic acid components, wherein the effectorprotein forms a complex with the one or more nucleic acid components andupon binding of the said complex to the locus of interest the effectorprotein induces the modification of the target locus of interest. In apreferred embodiment, the modification is the introduction of a strandbreak.

In a preferred embodiment, the target locus of interest comprises DNA.

In such methods the target locus of interest may be comprised within aDNA molecule or within an RNA molecule. In a preferred embodiment, thetarget locus of interest comprises RNA.

In such methods the target locus of interest may be comprised in a DNAmolecule within a cell. The cell may be a prokaryotic cell or aeukaryotic cell. The cell may be a mammalian cell. The mammalian cellmany be a non-human mammal, e.g., primate, bovine, ovine, porcine,canine, rodent, Leporidae such as monkey, cow, sheep, pig, dog, rabbit,rat or mouse cell. The cell may be a non-mammalian eukaryotic cell suchas poultry bird (e.g., chicken), vertebrate fish (e.g., salmon) orshellfish (e.g., oyster, claim, lobster, shrimp) cell. The cell may alsobe a plant cell. The plant cell may be of a monocot or dicot or of acrop or grain plant such as cassava, com, sorghum, soybean, wheat, oator rice. The plant cell may also be of an algae, tree or productionplant, fruit or vegetable (e.g., trees such as citrus trees, e.g.,orange, grapefruit or lemon trees; peach or nectarine trees; apple orpear trees; nut trees such as almond or walnut or pistachio trees;nightshade plants; plants of the genus Brassica; plants of the genusLactuca; plants of the genus Spinacia; plants of the genus Capsicum;cotton, tobacco, asparagus, carrot, cabbage, broccoli, cauliflower,tomato, eggplant, pepper, lettuce, spinach, strawberry, blueberry,raspberry, blackberry, grape, coffee, cocoa, etc).

In any of the described methods the target locus of interest may be agenomic or epigenomic locus of interest. In any of the described methodsthe complex may be delivered with multiple guides for multiplexed use.In any of the described methods more than one protein(s) may be used.

In preferred embodiments of the invention, biochemical or in vitro or invivo cleavage of sequences associated with or at a target locus ofinterest results without a putative transactivating crRNA (tracr RNA)sequence, e.g. cleavage by an AacC2c1 or C2c3 effector protein. In otherembodiments of the invention, cleavage may result with a putativetransactivating crRNA (tracr RNA) sequence, e.g. cleavage by otherCRISPR family effector proteins.

In any of the described methods the effector protein (e.g., C2c1 orC2c3) and nucleic acid components may be provided via one or morepolynucleotide molecules encoding the protein and/or nucleic acidcomponent(s), and wherein the one or more polynucleotide molecules areoperably configured to express the protein and/or the nucleic acidcomponent(s). The one or more polynucleotide molecules may comprise oneor more regulatory elements operably configured to express the proteinand/or the nucleic acid component(s). The one or more polynucleotidemolecules may be comprised within one or more vectors. The inventioncomprehends such polynucleotide molecule(s), for instance suchpolynucleotide molecules operably configured to express the proteinand/or the nucleic acid component(s), as well as such vector(s).

The invention also provides an engineered CRISPR protein, complex,composition, system, vector, cell or cell line according to theinvention for use in modifying a locus of interest in a cell. Saidmodifying preferably comprises contacting the cell with any of theabove-described compositions or any of the above-described systems. Theinvention also provides a use of an engineered CRISPR protein, complex,composition, system, vector, cell or cell line according to theinvention in the preparation of a medicament for modifying a locus ofinterest in a cell.

The invention also provides an engineered CRISPR protein, complex,composition, system, vector, cell or cell line according to theinvention for use in modifying a locus of interest in a cell. Theinvention also provides a use of an engineered CRISPR protein, complex,composition, system, vector, cell or cell line according to theinvention in the preparation of a medicament for modifying a locus ofinterest in a cell. Said modifying preferably comprises contacting thecell with any of the above-described compositions or any of theabove-described systems.

The invention also provides a method of modifying a target locus ofinterest, the method comprising delivering to said locus a non-naturallyoccurring or engineered composition comprising a C2c2 loci effectorprotein and one or more nucleic acid components, wherein the C2c2effector protein forms a complex with the one or more nucleic acidcomponents and upon binding of the said complex to the locus of interestthe effector protein induces the modification of the target locus ofinterest. In a preferred embodiment, the modification is theintroduction of a strand break.

In such methods the target locus of interest may be comprised in a DNAmolecule in vitro. In such methods the target locus of interest may becomprised in a DNA molecule within a cell. Preferably, in such methodsthe target locus of interest may be comprised in a RNA molecule invitro. Also preferably, in such methods the target locus of interest maybe comprised in a RNA molecule within a cell. The cell may be aprokaryotic cell or a eukaryotic cell. The cell may be a mammalian cell.The cell may be a rodent cell. The cell may be a mouse cell.

The invention also provides an engineered CRISPR protein, complex,composition, system, vector, cell or cell line according to theinvention for use in modifying a locus of interest in a cell. Saidmodifying preferably comprises contacting the cell with any of theabove-described compositions or any of the above-described systems. Theinvention also provides a use of an engineered CRISPR protein, complex,composition, system, vector, cell or cell line according to theinvention in the preparation of a medicament for modifying a locus ofinterest in a cell.

The invention also provides an engineered CRISPR protein, complex,composition, system, vector, cell or cell line according to theinvention for use in modifying a locus of interest in a cell. Theinvention also provides a use of an engineered CRISPR protein, complex,composition, system, vector, cell or cell line according to theinvention in the preparation of a medicament for modifying a locus ofinterest in a cell. Said modifying preferably comprises contacting thecell with any of the above-described compositions or any of theabove-described systems.

In any of the described methods the target locus of interest may be agenomic or epigenomic locus of interest. In any of the described methodsthe complex may be delivered with multiple guides for multiplexed use.In any of the described methods more than one protein(s) may be used.

In further aspects of the invention the nucleic acid components maycomprise a putative CRISPR RNA (crRNA) sequence and/or a putativetrans-activating crRNA (tracr RNA) sequence. In certain embodiments,cleavage such as biochemical or in vitro cleavage or cleavage in cells,can result without a putative transactivating crRNA (tracr RNA)sequence. In other embodiments, cleavage such as biochemical or in vitrocleavage or cleavage in cells, can result with a putativetransactivating crRNA (tracr RNA) sequence.

In certain embodiments, where the effector protein is a Type VCRISPR-Cas loci effector protein, such as a C2c1 loci effector proteinor a C2c3 loci effector protein, preferably a C2c1 loci effectorprotein, the nucleic acid components may comprise a putative CRISPR RNA(crRNA) sequence and a putative trans-activating crRNA (tracr RNA)sequence.

In further aspects of the invention the nucleic acid components maycomprise a putative CRISPR RNA (crRNA) sequence and not comprise anyputative trans-activating crRNA (tracr RNA) sequence. Withoutlimitation, the Applicants hypothesize that in such instances, thepre-crRNA may comprise secondary structure that is sufficient forprocessing to yield the mature crRNA as well as crRNA loading onto theeffector protein. By means of example and not limitation, such secondarystructure may comprise, consist essentially of or consist of a stem loopwithin the pre-crRNA, more particularly within the direct repeat.

In certain embodiments, where the effector protein is a Type VICRISPR-Cas loci effector protein, such as a C2c2 loci effector protein,the nucleic acid components may comprise a putative CRISPR RNA (crRNA)sequence and not comprise any putative trans-activating crRNA (tracrRNA) sequence.

In any of the described methods the effector protein and nucleic acidcomponents may be provided via one or more polynucleotide moleculesencoding the protein and/or nucleic acid component(s), and wherein theone or more polynucleotide molecules are operably configured to expressthe protein and/or the nucleic acid component(s). The one or morepolynucleotide molecules may comprise one or more regulatory elementsoperably configured to express the protein and/or the nucleic acidcomponent(s). The one or more polynucleotide molecules may be comprisedwithin one or more vectors. In any of the described methods the targetlocus of interest may be a genomic or epigenomic locus of interest. Inany of the described methods the complex may be delivered with multipleguides for multiplexed use. In any of the described methods more thanone protein(s) may be used.

In any of the described methods the strand break may be a single strandbreak or a double strand break.

Regulatory elements may comprise inducible promotors. Polynucleotidesand/or vector systems may comprise inducible systems.

In any of the described methods the one or more polynucleotide moleculesmay be comprised in a delivery system, or the one or more vectors may becomprised in a delivery system.

In any of the described methods the non-naturally occurring orengineered composition may be delivered via liposomes, particlesincluding nanoparticles, exosomes, microvesicles, a gene-gun or one ormore viral vectors, e.g., nucleic acid molecule or viral vectors.

The invention also provides a non-naturally occurring or engineeredcomposition which is a composition having the characteristics asdiscussed herein or defined in any of the herein described methods.

The invention also provides an engineered CRISPR protein, complex,composition, system, vector, cell or cell line according to theinvention for use in therapy.

In certain embodiments, the invention thus provides a non-naturallyoccurring or engineered composition, such as particularly a compositioncapable of or configured to modify a target locus of interest, saidcomposition comprising a Type V CRISPR-Cas loci effector protein and oneor more nucleic acid components, wherein the effector protein forms acomplex with the one or more nucleic acid components and upon binding ofthe said complex to the locus of interest the effector protein inducesthe modification of the target locus of interest. In certainembodiments, the effector protein may be encoded by a subtype V-ACRISPR-Cas loci or a subtype V-B CRISPR-Cas loci or a subtype V-CCRISPR-Cas loci. In certain embodiments, the effector protein may be aC2c1 loci effector protein or a C2c3 loci effector protein.

In certain embodiments, the invention thus provides a non-naturallyoccurring or engineered composition, such as particularly a compositioncapable of or configured to modify a target locus of interest, saidcomposition comprising a Type VI CRISPR-Cas loci effector protein andone or more nucleic acid components, wherein the effector protein formsa complex with the one or more nucleic acid components and upon bindingof the said complex to the locus of interest the effector proteininduces the modification of the target locus of interest. In certainembodiments, the effector protein may be a C2c2 loci effector protein.

The invention also provides in a further aspect a non-naturallyoccurring or engineered composition, such as particularly a compositioncapable of or configured to modify a target locus of interest, saidcomposition comprising: (a) a guide RNA molecule (or a combination ofguide RNA molecules, e.g., a first guide RNA molecule and a second guideRNA molecule) or a nucleic acid encoding the guide RNA molecule (or oneor more nucleic acids encoding the combination of guide RNA molecules);(b) a Type V CRISPR-Cas loci effector protein or a nucleic acid encodingthe Type V CRISPR-Cas loci effector protein. In certain embodiments, theeffector protein may be encoded by a subtype V-A CRISPR-Cas loci or asubtype V-B CRISPR-Cas loci or a subtype V-C CRISPR-Cas loci. In certainembodiments, the effector protein may be a C2c1 loci effector protein ora C2c3 loci effector protein.

The invention also provides in a further aspect a non-naturallyoccurring or engineered composition, such as particularly a compositioncapable of or configured to modify a target locus of interest, saidcomposition comprising: (a) a guide RNA molecule (or a combination ofguide RNA molecules, e.g., a first guide RNA molecule and a second guideRNA molecule) or a nucleic acid encoding the guide RNA molecule (or oneor more nucleic acids encoding the combination of guide RNA molecules);(b) a Type VI CRISPR-Cas loci effector protein or a nucleic acidencoding the Type VI CRISPR-Cas loci effector protein. In certainembodiments, the effector protein may be a C2c2 loci effector protein.

The invention also provides in a further aspect a non-naturallyoccurring or engineered composition comprising: (I.) one or moreCRISPR-Cas system polynucleotide sequences comprising (a) a guidesequence capable of hybridizing to a target sequence in a polynucleotidelocus, (b) a tracr mate sequence, and (c) a tracrRNA sequence, and (II.)a second polynucleotide sequence encoding a Type V CRISPR-Cas locieffector protein, wherein when transcribed, the tracr mate sequencehybridizes to the tracrRNA sequence and the guide sequence directssequence-specific binding of a CRISPR complex to the target sequence,and wherein the CRISPR complex comprises the Type V CRISPR-Cas locieffector protein complexed with (1) the guide sequence that ishybridized to the target sequence, and (2) the tracr mate sequence thatis hybridized to the tracrRNA sequence. In certain embodiments, theeffector protein may be encoded by a subtype V-A CRISPR-Cas loci or asubtype V-B CRISPR-Cas loci or a subtype V-C CRISPR-Cas loci. In certainembodiments, the effector protein may be a C2c1 loci effector protein ora C2c3 loci effector protein.

The invention also provides in a further aspect a non-naturallyoccurring or engineered composition comprising: (I.) one or moreCRISPR-Cas system polynucleotide sequences comprising (a) a guidesequence capable of hybridizing to a target sequence in a polynucleotidelocus, (b) a tracr mate sequence, and (c) a tracrRNA sequence, and (II.)a second polynucleotide sequence encoding a Type VI CRISPR-Cas locieffector protein, wherein when transcribed, the tracr mate sequencehybridizes to the tracrRNA sequence and the guide sequence directssequence-specific binding of a CRISPR complex to the target sequence,and wherein the CRISPR complex comprises the Type VI CRISPR-Cas locieffector protein complexed with (1) the guide sequence that ishybridized to the target sequence, and (2) the tracr mate sequence thatis hybridized to the tracrRNA sequence. In certain embodiments, theeffector protein may be a C2c2 loci effector protein.

In certain embodiments, a tracrRNA may not be required. Hence, theinvention also provides in certain embodiments a non-naturally occurringor engineered composition comprising: (I.) one or more CRISPR-Cas systempolynucleotide sequences comprising (a) a guide sequence capable ofhybridizing to a target sequence in a polynucleotide locus, and (b) adirect repeat sequence, and (II.) a second polynucleotide sequenceencoding a Type V or Type VI CRISPR-Cas loci effector protein, whereinwhen transcribed, the guide sequence directs sequence-specific bindingof a CRISPR complex to the target sequence, and wherein the CRISPRcomplex comprises the Type V or Type VI CRISPR-Cas loci effector proteincomplexed with (1) the guide sequence that is hybridized to the targetsequence, and (2) the direct repeat sequence. In certain embodiments,the Type V effector protein may be encoded by a subtype V-A CRISPR-Casloci or a subtype V-B CRISPR-Cas loci or a subtype V-C CRISPR-Cas loci.In certain embodiments, the effector protein may be a C2c1 loci effectorprotein or a C2c3 loci effector protein. Preferably, the effectorprotein may be a Type VI CRISPR-Cas loci effector protein. Morepreferably, the effector protein may be a C2c2 loci effector protein.Without limitation, the Applicants hypothesise that in such instances,the direct repeat sequence may comprise secondary structure that issufficient for crRNA loading onto the effector protein. By means ofexample and not limitation, such secondary structure may comprise,consist essentially of or consist of a stem loop within the directrepeat.

The invention also provides a vector system comprising one or morevectors, the one or more vectors comprising one or more polynucleotidemolecules encoding components of a non-naturally occurring or engineeredcomposition which is a composition having the characteristics as definedin any of the herein described methods.

The invention also provides a delivery system comprising one or morevectors or one or more polynucleotide molecules, the one or more vectorsor polynucleotide molecules comprising one or more polynucleotidemolecules encoding components of a non-naturally occurring or engineeredcomposition which is a composition having the characteristics discussedherein or as defined in any of the herein described methods.

The invention also provides a non-naturally occurring or engineeredcomposition, or one or more polynucleotides encoding components of saidcomposition, or vector or delivery systems comprising one or morepolynucleotides encoding components of said composition for use in atherapeutic method of treatment. The therapeutic method of treatment maycomprise gene or genome editing, or gene therapy.

The invention also provides an engineered CRISPR protein, complex,composition, system, vector, cell or cell line according to theinvention for use in the treatment of a disease, disorder or infectionin an individual in need thereof. The disease, disorder or infection maycomprise a viral infection. The viral infection may be HBV. Theinvention also provides a use of an engineered CRISPR protein, complex,composition, system, vector, cell or cell line according to theinvention in the preparation of a medicament for the treatment of adisease, disorder or infection in an individual in need thereof. Thedisease, disorder or infection may comprise a viral infection.

The invention also encompasses computational methods and algorithms topredict new Class 2 CRISPR-Cas systems and identify the componentstherein.

The invention also provides for methods and compositions wherein one ormore amino acid residues of the effector protein may be modified e.g. anengineered or non-naturally-occurring effector protein or C2c1 or C2c3.In an embodiment, the modification may comprise mutation of one or moreamino acid residues of the effector protein. The one or more mutationsmay be in one or more catalytically active domains of the effectorprotein. The effector protein may have reduced or abolished nucleaseactivity compared with an effector protein lacking said one or moremutations. The effector protein may not direct cleavage of one or otherDNA or RNA strand at the target locus of interest. The effector proteinmay not direct cleavage of either DNA or RNA strand at the target locusof interest. In a preferred embodiment, the one or more mutations maycomprise two mutations. In a preferred embodiment the one or more aminoacid residues are modified in a C2c1 or C2c3 effector protein, e.g. anengineered or non-naturally-occurring effector protein or C2c1 or C2c3.

The invention also provides for the one or more mutations or the two ormore mutations to be in a catalytically active domain of the effectorprotein comprising a RuvC domain. In some embodiments of the inventionthe RuvC domain may comprise a RuvCI, RuvCII or RuvCIII domain, or acatalytically active domain which is homologous to a RuvCI, RuvCII orRuvCIII domain etc or to any relevant domain as described in any of theherein described methods. In certain embodiments, the one or moremutations or the two or more mutations may be in a catalytically activedomain of the effector protein comprising a HEPN domain, or acatalytically active domain which is homologous to a HEPN domain. Theeffector protein may comprise one or more heterologous functionaldomains. The one or more heterologous functional domains may compriseone or more nuclear localization signal (NLS) domains. The one or moreheterologous functional domains may comprise at least two or more NLSdomains. The one or more NLS domain(s) may be positioned at or near orin proximity to a terminus of the effector protein (e.g., C2c1 or C2c3)and if two or more NLSs, each of the two may be positioned at or near orin proximity to a terminus of the effector protein (e.g., C2c1 or C2c3).The one or more heterologous functional domains may comprise one or moretranscriptional activation domains. In a preferred embodiment thetranscriptional activation domain may comprise VP64. The one or moreheterologous functional domains may comprise one or more transcriptionalrepression domains. In a preferred embodiment the transcriptionalrepression domain comprises a KRAB domain or a SID domain (e.g. SID4X).The one or more heterologous functional domains may comprise one or morenuclease domains. In a preferred embodiment a nuclease domain comprisesFok1.

The invention also provides for the one or more heterologous functionaldomains to have one or more of the following activities: methylaseactivity, demethylase activity, transcription activation activity,transcription repression activity, transcription release factoractivity, histone modification activity, nuclease activity,single-strand RNA cleavage activity, double-strand RNA cleavageactivity, single-strand DNA cleavage activity, double-strand DNAcleavage activity and nucleic acid binding activity. At least one ormore heterologous functional domains may be at or near theamino-terminus of the effector protein and/or wherein at least one ormore heterologous functional domains is at or near the carboxy-terminusof the effector protein. The one or more heterologous functional domainsmay be fused to the effector protein. The one or more heterologousfunctional domains may be tethered to the effector protein. The one ormore heterologous functional domains may be linked to the effectorprotein by a linker moiety.

The invention also provides for the effector protein comprising aneffector protein from an organism from a genus comprising Streptococcus,Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia,Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta,Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter,Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridium,Leptotrichia, Francisella, Legionella, Alicyclobacillus,Methanomethylophilus, Porphyromonas, Prevotella, Bacteroidetes,Helcococcus, Leptospira, Desulfovibrio, Desulfonatronum, Opitutaceae,Tuberibacillus, Bacillus, Brevibacillus, Methylobacterium orAcidaminococcus. The effector protein may comprise a chimeric effectorprotein comprising a first fragment from a first effector proteinortholog and a second fragment from a second effector protein ortholog,and wherein the first and second effector protein orthologs aredifferent. At least one of the first and second effector proteinorthologs may comprise an effector protein from an organism comprisingStreptococcus, Campylobacter, Nitratifractor, Staphylococcus,Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum,Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium,Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae,Clostridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus,Methanomethylophilus, Porphyromonas, Prevotella, Bacteroidetes,Helcococcus, Leptospira, Desulfovibrio, Desulfonatronum, Opitutaceae,Tuberibacillus, Bacillus, Brevibacillus, Methylobacterium orAcidaminococcus.

In certain embodiments, the effector protein, particularly a Type V locieffector protein, more particularly a Type V-B loci effector protein,even more particularly a C2c1p, may originate from, may be isolated fromor may be derived from a bacterial species belonging to the taxaBacilli, Verrucomicrobia, alpha-proteobacteria or delta-proteobacteria.In certain embodiments, the effector protein, particularly a Type V locieffector protein, more particularly a Type V-B loci effector protein,even more particularly a C2c1p, may originate from, may be isolated fromor may be derived from a bacterial species belonging to a genus selectedfrom the group consisting of Alicyclobacillus, Desulfovibrio,Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacillus,Desulfatirhabdium, Citrobacter, and Methylobacterium. In certainembodiments, the effector protein, particularly a Type V loci effectorprotein, more particularly a Type V-B loci effector protein, even moreparticularly a C2c1p, may originate, may be isolated or may be derivedfrom a bacterial species selected from the group consisting ofAlicyclobacillus acidoterrestris (e.g., ATCC 49025), Alicyclobacilluscontaminans (e.g., DSM 17975), Desulfovibrio inopinatus (e.g., DSM10711), Desulfonatronum thiodismutans (e.g., strain MLF-1), Opitutaceaebacterium TAV5, Tuberibacillus calidus (e.g., DSM 17572), Bacillusthermoamylovorans (e.g., strain B4166), Brevibacillus sp. CF112,Bacillus sp. NSP2.1, Desulfatirhabdium butyrativorans (e.g., DSM 18734),Alicyclobacillus herbarius (e.g., DSM 13609), Citrobacter freundii(e.g., ATCC 8090), Brevibacillus agri (e.g., BAB-2500), Methylobacteriumnodulans (e.g., ORS 2060). In certain embodiments, the effector protein,particularly a Type V loci effector protein, more particularly a TypeV-B loci effector protein, even more particularly a C2c1p, mayoriginate, may be isolated or may be derived from a bacterial speciesselected from the group consisting of the bacterial species listed inthe Table in FIG. 41A-B.

In certain embodiments, the effector protein, particularly a Type V locieffector protein, more particularly a Type V-B loci effector protein,even more particularly a C2c1p, may comprise, consist essentially of orconsist of an amino acid sequence selected from the group consisting ofamino acid sequences shown in the multiple sequence alignment in FIG.13D-1-H-2.

In certain embodiments, a Type V-B locus as intended herein may encode aCas1-Cas4 fusion, Cas2, and the C2c1p effector protein. In certainembodiments, a Type V-B locus as intended herein may be adjacent to aCRISPR array. See FIG. 9 and FIG. 41A-B for illustration ofrepresentative Type V-B loci organization.

In certain embodiments, a Cas1 protein encoded by a Type V-B locus asintended herein may cluster with Type I-U system. See FIGS. 10A and 10Band FIG. 10C-1-W illustrating a Cas1 tree including Cas1 encoded byrepresentative Type V-B loci.

In certain embodiments, the effector protein, particularly a Type V locieffector protein, more particularly a Type V-B loci effector protein,even more particularly a C2c1p, such as a native C2c1p, may be about1100 to about 1500 amino acids long, e.g., about 1100 to about 1200amino acids long, or about 1200 to about 1300 amino acids long, or about1300 to about 1400 amino acids long, or about 1400 to about 1500 aminoacids long, e.g., about 1100, about 1200, about 1300, about 1400 orabout 1500 amino acids long.

In certain embodiments, the effector protein, particularly a Type V locieffector protein, more particularly a Type V-B loci effector protein,even more particularly a C2c1p, and preferably the C-terminal portion ofsaid effector protein, comprises the three catalytic motifs of theRuvC-like nuclease (i.e., RuvCI, RuvCII and RuvCIII). In certainembodiments, said effector protein, and preferably the C-terminalportion of said effector protein, may further comprise a regioncorresponding to the bridge helix (also known as arginine-rich cluster)that in Cas9 protein is involved in crRNA-binding. In certainembodiments, said effector protein, and preferably the C-terminalportion of said effector protein, may further comprise a Zn fingerregion, which may be inactive (i.e., which does not bind zinc, e.g., inwhich the Zn-binding cysteine residue(s) are missing). In certainembodiments, said effector protein, and preferably the C-terminalportion of said effector protein, may comprise the three catalyticmotifs of the RuvC-like nuclease (i.e., RuvCI, RuvCII and RuvCIII), theregion corresponding to the bridge helix, and the Zn finger region,preferably in the following order, from N to C terminus: RuvCI-bridgehelix-RuvCII-Zinc finger-RuvCIII. See FIG. 11, FIG. 12-1-2 and FIGS.13A-1-A-2 and 13C-1-C-2 for illustration of representative Type V-Beffector proteins domain architecture.

In certain embodiments, Type V-B loci as intended herein may compriseCRISPR repeats between 30 and 40 bp long, more typically between 34 and38 bp long, even more typically between 36 and 37 bp long, e.g., 30, 31,32, 33, 34, 35, 36, 37, 38, 39, or 40 bp long.

In certain embodiments, the effector protein, particularly a Type V locieffector protein, more particularly a Type V-C loci effector protein,even more particularly a C2c3p, may originate, may be isolated or may bederived from a bacterial metagenome selected from the group consistingof the bacterial metagenomes listed in the Table in FIG. 43A-B.

In certain embodiments, the effector protein, particularly a Type V locieffector protein, more particularly a Type V-C loci effector protein,even more particularly a C2c3p, may comprise, consist essentially of orconsist of an amino acid sequence selected from the group consisting ofamino acid sequences shown in the multiple sequence alignment in FIG.13I-1-I-4.

In certain embodiments, a Type V-C locus as intended herein may encodeCas1 and the C2c3p effector protein. See FIG. 14 and FIG. 43A-B forillustration of representative Type V-C loci organization.

In certain embodiments, a Cas1 protein encoded by a Type V-C locus asintended herein may cluster with Type I-B system. See FIGS. 10A and 10Band FIG. 10C-1-W illustrating a Cas1 tree including Cas1 encoded byrepresentative Type V-C loci.

In certain embodiments, the effector protein, particularly a Type V locieffector protein, more particularly a Type V-C loci effector protein,even more particularly a C2c3p, such as a native C2c3p, may be about1100 to about 1500 amino acids long, e.g., about 1100 to about 1200amino acids long, or about 1200 to about 1300 amino acids long, or about1300 to about 1400 amino acids long, or about 1400 to about 1500 aminoacids long, e.g., about 1100, about 1200, about 1300, about 1400 orabout 1500 amino acids long, or at least about 1100, at least about1200, at least about 1300, at least about 1400 or at least about 1500amino acids long.

In certain embodiments, the effector protein, particularly a Type V locieffector protein, more particularly a Type V-C loci effector protein,even more particularly a C2c3p, and preferably the C-terminal portion ofsaid effector protein, comprises the three catalytic motifs of theRuvC-like nuclease (i.e., RuvCI, RuvCII and RuvCIII). In certainembodiments, said effector protein, and preferably the C-terminalportion of said effector protein, may further comprise a regioncorresponding to the bridge helix (also known as arginine-rich cluster)that in Cas9 protein is involved in crRNA-binding. In certainembodiments, said effector protein, and preferably the C-terminalportion of said effector protein, may further comprise a Zn fingerregion. Preferably, the Zn-binding cysteine residue(s) may be conservedin C2c3p. In certain embodiments, said effector protein, and preferablythe C-terminal portion of said effector protein, may comprise the threecatalytic motifs of the RuvC-like nuclease (i.e., RuvCI, RuvCII andRuvCIII), the region corresponding to the bridge helix, and the Znfinger region, preferably in the following order, from N to C terminus:RuvCI-bridge helix-RuvCII-Zinc finger-RuvCIII. See FIGS. 13A-1-A-2 and13C-1-C-2 for illustration of representative Type V-C effector proteinsdomain architecture.

In certain embodiments, Type V-C loci as intended herein may compriseCRISPR repeats between 20 and 30 bp long, more typically between 22 and27 bp long, yet more typically 25 bp long, e.g., 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 bp long.

In certain embodiments, the effector protein, particularly a Type VIloci effector protein, more particularly a C2c2p, may originate from,may be isolated from, or may be derived from a bacterial speciesbelonging to the taxa alpha-proteobacteria, Bacilli, Clostridia,Fusobacteria and Bacteroidetes. In certain embodiments, the effectorprotein, particularly a Type VI loci effector protein, more particularlya C2c2p, may originate from, may be isolated from, or may be derivedfrom a bacterial species belonging to a genus selected from the groupconsisting of Lachnospiraceae, Clostridium, Carnobacterium,Paludibacter, Listeria, Leptotrichia, and Rhodobacter. In certainembodiments, the effector protein, particularly a Type VI loci effectorprotein, more particularly a C2c2p may originate from, may be isolatedfrom or may be derived from a bacterial species selected from the groupconsisting of Lachnospiraceae bacterium MA2020, Lachnospiraceaebacterium NK4A179, Clostridium aminophilum (e.g., DSM 10710),Lachnospiraceae bacterium NK4A144, Carnobacterium gallinarum (e.g., DSM4847 strain MT44), Paludibacter propionicigenes (e.g., WB4), Listeriaseeligeri (e.g., serovar ½b str. SLCC3954), Listeria weihenstephanensis(e.g., FSL R9-0317 c4), Listeria newyorkensis (e.g., strain FSLM6-0635), Leptotrichia wadei (e.g., F0279), Leptotrichia buccalis (e.g.,DSM 1135), Leptotrichia sp. Oral taxon 225 (e.g., str. F0581),Leptotrichia sp. Oral taxon 879 (e.g., strain F0557), Leptotrichiashahii (e.g., DSM 19757), Rhodobacter capsulatus (e.g., SB 1003, R121,or DE442). In certain embodiments, the effector protein, particularly aType VI loci effector protein, more particularly a C2c2p may originatefrom, may be isolated from or may be derived from a bacterial speciesselected from the group consisting of the bacterial species listed inthe Table in FIG. 42A-B.

In certain embodiments, the effector protein, particularly a Type VIloci effector protein, more particularly a C2c2p, may comprise, consistessentially of or consist of an amino acid sequence selected from thegroup consisting of amino acid sequences shown in the multiple sequencealignment in FIG. 13J-1-N-4.

In certain embodiments, a Type VI locus as intended herein may encodeCas1, Cas2, and the C2c2p effector protein. In certain embodiments, aType V-C locus as intended herein may comprise a CRISPR array. Incertain embodiments, a Type V-C locus as intended herein may comprisethe c2c2 gene and a CRISPR array, and not comprise cas1 and cas2 genes.See FIG. 15 and FIG. 42A-B for illustration of representative Type VIloci organization.

In certain embodiments, a Cas1 protein encoded by a Type VI locus asintended herein may cluster within the Type II subtree along with asmall Type III-A branch, or within Type III-A system. See FIGS. 10A and10B and FIG. 10C-1-W illustrating a Cas1 tree including Cas1 encoded byrepresentative Type VI loci.

In certain embodiments, the effector protein, particularly a Type VIloci effector protein, more particularly a C2c2p, such as a nativeC2c2p, may be about 1000 to about 1500 amino acids long, such as about1100 to about 1400 amino acids long, e.g., about 1000 to about 1100,about 1100 to about 1200 amino acids long, or about 1200 to about 1300amino acids long, or about 1300 to about 1400 amino acids long, or about1400 to about 1500 amino acids long, e.g., about 1000, about 1100, about1200, about 1300, about 1400 or about 1500 amino acids long.

In certain embodiments, the effector protein, particularly a Type VIloci effector protein, more particularly a C2c2p, comprises at least oneand preferably at least two, such as more preferably exactly two,conserved RxxxxH motifs. Catalytic RxxxxH motifs are characteristic ofHEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding) domains.Hence, in certain embodiments, the effector protein, particularly a TypeVI loci effector protein, more particularly a C2c2p, comprises at leastone and preferably at least two, such as more preferably exactly two,HEPN domains. See FIG. 11 and FIG. 13B for illustration ofrepresentative Type VI effector proteins domain architecture. In certainembodiments, the HEPN domains may possess RNAse activity. In otherembodiments, the HEPN domains may possess DNAse activity.

In certain embodiments, Type VI loci as intended herein may compriseCRISPR repeats between 30 and 40 bp long, more typically between 35 and39 bp long, e.g., 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 bp long.

In certain embodiments, a protospacer adjacent motif (PAM) or PAM-likemotif directs binding of the effector protein complex as disclosedherein to the target locus of interest. In some embodiments, the PAM maybe a 5′ PAM (i.e., located upstream of the 5′ end of the protospacer).In other embodiments, the PAM may be a 3′ PAM (i.e., located downstreamof the 5′ end of the protospacer).

In a preferred embodiment, the effector protein, particularly a Type Vloci effector protein, more particularly a Type V-B loci effectorprotein, even more particularly a C2c1p, may recognize a 5′ PAM. Incertain embodiments, the effector protein, particularly a Type V locieffector protein, more particularly a Type V-B loci effector protein,even more particularly a C2c1p, may recognize a 5′ PAM which is 5′ TTNor 5′ ATTN, where N is A, C, G or T. In certain preferred embodiments,the effector protein may be Alicyclobacillus acidoterrestris C2c1p, morepreferably Alicyclobacillus acidoterrestris ATCC 49025 C2c1p, and the 5′PAM is 5′ TTN, where N is A, C, G or T, more preferably where N is A, Gor T. In other preferred embodiments, the effector protein is Bacillusthermoamylovorans C2c1p, more preferably Bacillus thermoamylovoransstrain B4166 C2c1p, and the 5′ PAM is 5′ ATTN, where N is A, C, G or T.

In certain embodiments, the CRISPR enzyme is engineered and can compriseone or more mutations that reduce or eliminate a nuclease activity.

Mutations can also be made at neighboring residues, e.g., at amino acidsnear those indicated above that participate in the nuclease activity. Insome embodiments, only the RuvC domain is inactivated, and in otherembodiments, another putative nuclease domain is inactivated, whereinthe effector protein complex functions as a nickase and cleaves only oneDNA strand. In some embodiments, two C2c1 or C2c3 variants (each adifferent nickase) are used to increase specificity, two nickasevariants are used to cleave DNA at a target (where both nickases cleavea DNA strand, while minimizing or eliminating off-target modificationswhere only one DNA strand is cleaved and subsequently repaired). Inpreferred embodiments the C2c1 or C2c3 effector protein cleavessequences associated with or at a target locus of interest as ahomodimer comprising two C2c1 or C2c3 effector protein molecules. In apreferred embodiment the homodimer may comprise two C2c1 or two C2c3effector protein molecules, or a mixture of C2c1 and C2c3, comprising adifferent mutation in their respective RuvC domains.

The invention contemplates methods of using two or more nickases, inparticular a dual or double nickase approach. In some aspects andembodiments, a single type C2c1 or C2c3 nickase may be delivered, forexample a modified C2c1 or C2c3 or a modified C2c1 or C2c3 nickase asdescribed herein. This results in the target DNA being bound by two C2c1or two C2c3 nickases, or a mixture of C2c1 and C2c3 nickases. Inaddition, it is also envisaged that different orthologs may be used,e.g, an C2c1 or C2c3 nickase on one strand (e.g., the coding strand) ofthe DNA and an ortholog on the non-coding or opposite DNA strand. Theortholog can be, but is not limited to, a Cas9 nickase such as a SaCas9nickase or a SpCas9 nickase. It may be advantageous to use two differentorthologs that require different PAMs and may also have different guiderequirements, thus allowing a greater deal of control for the user. Incertain embodiments, DNA cleavage will involve at least four types ofnickases, wherein each type is guided to a different sequence of targetDNA, wherein each pair introduces a first nick into one DNA strand andthe second introduces a nick into the second DNA strand. In suchmethods, at least two pairs of single stranded breaks are introducedinto the target DNA wherein upon introduction of first and second pairsof single-strand breaks, target sequences between the first and secondpairs of single-strand breaks are excised. In certain embodiments, oneor both of the orthologs is controllable, i.e. inducible.

In certain embodiments of the invention, the guide RNA or mature crRNAcomprises, consists essentially of, or consists of a direct repeatsequence and a guide sequence or spacer sequence. In certainembodiments, the guide RNA or mature crRNA comprises, consistsessentially of, or consists of a direct repeat sequence linked to aguide sequence or spacer sequence. In certain embodiments the guide RNAor mature crRNA comprises 19 nts of partial direct repeat followed by23-25 nt of guide sequence or spacer sequence. In certain embodiments,the effector protein is a C2c1 or C2c3 effector protein and requires atleast 16 nt of guide sequence to achieve detectable DNA cleavage and aminimum of 17 nt of guide sequence to achieve efficient DNA cleavage invitro. In certain embodiments, the direct repeat sequence is locatedupstream (i.e., 5′) from the guide sequence or spacer sequence. In apreferred embodiment the seed sequence (i.e. the sequence essentialcritical for recognition and/or hybridization to the sequence at thetarget locus) of the C2c1 or C2c3 guide RNA is approximately within thefirst 5 nt on the 5′ end of the guide sequence or spacer sequence.

In preferred embodiments of the invention, the mature crRNA comprises astem loop or an optimized stem loop structure or an optimized secondarystructure. In preferred embodiments the mature crRNA comprises a stemloop or an optimized stem loop structure in the direct repeat sequence,wherein the stem loop or optimized stem loop structure is important forcleavage activity. In certain embodiments, the mature crRNA preferablycomprises a single stem loop. In certain embodiments, the direct repeatsequence preferably comprises a single stem loop. In certainembodiments, the cleavage activity of the effector protein complex ismodified by introducing mutations that affect the stem loop RNA duplexstructure. In preferred embodiments, mutations which maintain the RNAduplex of the stem loop may be introduced, whereby the cleavage activityof the effector protein complex is maintained. In other preferredembodiments, mutations which disrupt the RNA duplex structure of thestem loop may be introduced, whereby the cleavage activity of theeffector protein complex is completely abolished.

The invention also provides for the nucleotide sequence encoding theeffector protein being codon optimized for expression in a eukaryote oreukaryotic cell in any of the herein described methods or compositions.In an embodiment of the invention, the codon optimized effector proteinis any C2c1 or C2c3 discussed herein and is codon optimized foroperability in a eukaryotic cell or organism, e.g., such cell ororganism as elsewhere herein mentioned, for instance, withoutlimitation, a yeast cell, or a mammalian cell or organism, including amouse cell, a rat cell, and a human cell or non-human eukaryoteorganism, e.g., plant.

In certain embodiments of the invention, at least one nuclearlocalization signal (NLS) is attached to the nucleic acid sequencesencoding the C2c1 or C2c3 effector proteins. In preferred embodiments atleast one or more C-terminal or N-terminal NLSs are attached (and hencenucleic acid molecule(s) coding for the C2c1 or C2c3 effector proteincan include coding for NLS(s) so that the expressed product has theNLS(s) attached or connected). In a preferred embodiment a C-terminalNLS is attached for optimal expression and nuclear targeting ineukaryotic cells, preferably human cells. In a preferred embodiment, thecodon optimized effector protein is C2c1 or C2c3 and the spacer lengthof the guide RNA is from 15 to 35 nt. In certain embodiments, the spacerlength of the guide RNA is at least 16 nucleotides, such as at least 17nucleotides. In certain embodiments, the spacer length is from 15 to 17nt, from 17 to 20 nt, from 20 to 24 nt, eg. 20, 21, 22, 23, or 24 nt,from 23 to 25 nt, e.g., 23, 24, or 25 nt, from 24 to 27 nt, from 27-30nt, from 30-35 nt, or 35 nt or longer. In certain embodiments of theinvention, the codon optimized effector protein is C2c1 or C2c3 and thedirect repeat length of the guide RNA is at least 16 nucleotides. Incertain embodiments, the codon optimized effector protein is C2c1 orC2c3 and the direct repeat length of the guide RNA is from 16 to 20 nt,e.g., 16, 17, 18, 19, or 20 nucleotides. In certain preferredembodiments, the direct repeat length of the guide RNA is 19nucleotides.

The invention also encompasses methods for delivering multiple nucleicacid components, wherein each nucleic acid component is specific for adifferent target locus of interest thereby modifying multiple targetloci of interest. The nucleic acid component of the complex may compriseone or more protein-binding RNA aptamers. The one or more aptamers maybe capable of binding a bacteriophage coat protein. The bacteriophagecoat protein may be selected from the group comprising Qβ, F2, GA, fr,JP501, MS2, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP,FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s and PRR1. Ina preferred embodiment the bacteriophage coat protein is MS2. Theinvention also provides for the nucleic acid component of the complexbeing 30 or more, 40 or more or 50 or more nucleotides in length.

The invention also encompasses the cells, components and/or systems ofthe present invention having trace amounts of cations present in thecells, components and/or systems. Advantageously, the cation ismagnesium, such as Mg²⁺. The cation may be present in a trace amount. Apreferred range may be about 1 mM to about 15 mM for the cation, whichis advantageously Mg²⁺. A preferred concentration may be about 1 mM forhuman based cells, components and/or systems and about 10 mM to about 15mM for bacteria based cells, components and/or systems. See, e.g.,Gasiunas et al., PNAS, published online Sep. 4, 2012,www.pnas.org/cgi/doi/10.1073/pnas.1208507109.

Accordingly, it is an object of the invention not to encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. § 112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product. It may be advantageous in thepractice of the invention to be in compliance with Art. 53(c) EPC andRule 28(b) and (c) EPC. Nothing herein is to be construed as a promise.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

In a further aspect, the invention provides a eukaryotic cell comprisinga modified target locus of interest, wherein the target locus ofinterest has been modified according to in any of the herein describedmethods. A further aspect provides a cell line of said cell. Anotheraspect provides a multicellular organism comprising one or more saidcells.

In certain embodiments, the modification of the target locus of interestmay result in: the eukaryotic cell comprising altered expression of atleast one gene product; the eukaryotic cell comprising alteredexpression of at least one gene product, wherein the expression of theat least one gene product is increased; the eukaryotic cell comprisingaltered expression of at least one gene product, wherein the expressionof the at least one gene product is decreased; or the eukaryotic cellcomprising an edited genome.

In certain embodiments, the eukaryotic cell may be a mammalian cell or ahuman cell.

In further embodiments, the non-naturally occurring or engineeredcompositions, the vector systems, or the delivery systems as describedin the present specification may be used for: site-specific geneknockout; site-specific genome editing; DNA sequence-specificinterference; or multiplexed genome engineering.

Also provided is a gene product from the cell, the cell line, or theorganism as described herein. In certain embodiments, the amount of geneproduct expressed may be greater than or less than the amount of geneproduct from a cell that does not have altered expression or editedgenome. In certain embodiments, the gene product may be altered incomparison with the gene product from a cell that does not have alteredexpression or edited genome.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIGS. 1A and 1B depicts a new classification of CRISPR-Cas systems.Class 1 includes multisubunit crRNA-effector complexes (Cascade) andClass 2 includes Single-subunit crRNA-effector complexes (Cas9-like).FIG. 1B provides another depiction of the new classification ofCRISPR-Cas systems.

FIG. 2 provides a molecular organization of CRISPR-Cas.

FIG. 3A-3D provides structures of Type I and III effector complexes:common architecture/common ancestry despite extensive sequencedivergence.

FIG. 4 shows CRISPR-Cas as a RNA recognition motif (RRM)-centeredsystem.

FIG. 5 shows Cas1 phylogeny where recombination of adaptation andcrRNA-effector modules show a major aspect of CRISPR-Cas evolution.

FIG. 6 shows a CRISPR-Cas census, specifically a distribution ofCRISPR-Cas types/subtypes among archaea and bacteria.

FIG. 7 depicts a pipeline for identifying Cas candidates.

FIGS. 8A and 8B depicts an organization of complete loci of Class 2CRISPR-Cas systems. The three subtypes of type II and subtypes V-A, V-Band V-C, and type VI are indicated. Subfamilies based on Cas1 are alsoindicated. The schematics include only the common genes represented ineach subtype; the additional genes present in some variants are omitted.The red rectangle shows the degenerate repeat. The gray arrows show thedirection of CRISPR array transcription. PreFran,Prevotella-Francisella. FIG. 8B provides another depiction of anorganization of complete loci of several Class 2 CRISPR-Cas systems.

FIG. 9 depicts C2c1 neighborhoods, i.e., genomic architecture of theC2c1 CRISPR-Cas loci. The number of repeats in CRISPR arrays isindicated. For each genomic contig, Genbank numeric ID and thecoordinates of the locus are indicated.

FIGS. 10A and 10B depict representations of a Cas1 tree. The tree inFIG. 10B was constructed from a multiple alignment of 1498 Cas1sequences which contained 304 phylogenetically informative positions.Branches, corresponding to Class 2 systems are highlighted: cyan, typeII; orange, subtype V-A; red, subtype V-B; brown, subtype V-C; purple,type VI. Insets show the expanded branches of the novel (sub)types. Thebootstrap support values are given as percentage points and shown onlyfor few relevant branches.

FIG. 10C-1-10W provide the complete Cas1 tree, which is schematicallyshown in FIG. 10B, in Newick format with species names and bootstrapsupport values. The tree was reconstructed by FastTree program (“−gamma−wag” options). A multiple alignment of Cas1 sequences was filtered withhomogeneity threshold of 0.1 and gap occurrence threshold of 0.5, priorto tree reconstruction.

FIG. 11 depicts a domain organization of class 2 families.

FIG. 12-1-12-2 depicts TnpB homology regions in Class 2 proteins. FIG.12-1-12-2 discloses SEQ ID NOS 202-384, respectively, in order ofappearance.

FIG. 13A-1-13N-4 provide another depiction of domain architectures andconserved motifs of the Class 2 effector proteins. FIG. 13A-1-A-2illustrates Types II and V: TnpB-derived nucleases. The top panel showsthe RuvC nuclease from Thermos thermophilus (PDB ID: 4EP5) with thecatalytic amino acid residues denoted. Underneath each domainarchitecture, an alignment of the conserved motifs in selectedrepresentatives of the respective protein family (a single sequence forRuvC) is shown. The catalytic residues are shown by white letters on ablack background; conserved hydrophobic residues are highlighted inyellow; conserved small residues are highlighted in green; in the bridgehelix alignment, positively charged residues are in red. Secondarystructure prediction is shown underneath the aligned sequences: Hdenotes α-helix and E denotes extended conformation (β-strand). Thepoorly conserved spacers between the alignment blocks are shown bynumbers. FIG. 13B illustrates Type VI: proteins containing two HEPNdomains, which may display RNAse activity. The top alignment blocksinclude selected HEPN domains described previously and the bottom blocksinclude the catalytic motifs from the type VI effector proteins. Thedesignations are as in FIG. 13A-1-A-2. FIG. 13C-1-C-2 shows the closesthomologs of the new type V effector proteins among thetransposon-encoded proteins: non-overlapping sets of homologs. FIG.13D-1-H-2 shows multiple alignment of C2c1 protein family. The alignmentwas built using MUSCLE program and modified manually on the basis oflocal PSI-BLAST pairwise alignments. Each sequence is labelled withGenBank Identifier (GI) number and systematic name of an organism.Secondary structure was predicted by Jpred and shown underneath thesequence which was used as a query (designations: H-alpha helix, E-betastrand). CONSENSUS was calculated for each alignment column by scalingthe sum-of-pairs score within the column between those of a homogeneouscolumn (the same residue in all aligned sequences) and a random columnwith homogeneity cutoff 0.8. Active site motifs of RuvC-like domain areshown below alignment. FIG. 13I-1-I-4 shows multiple alignment of C2c3protein family. The alignment was built using MUSCLE program. Eachsequence is labelled with local assigned number and the Genbank ID formetagenomics contig coding for respective C2c3 protein. Secondarystructure was predicted by Jpred and shown underneath the alignment(designations: H-alpha helix, E-beta strand). CONSENSUS was calculatedfor each alignment column by scaling the sum-of-pairs score within thecolumn between those of a homogeneous column (the same residue in allaligned sequences) and a random column with homogeneity cutoff 0.8.Active site motifs of RuvC-like domain are shown below alignment for theC-terminal domain. FIG. 13J-1-N-4 shows multiple alignment of C2c2protein family. The alignment was built using MUSCLE program andmodified manually on the basis of local PSIBLAST pairwise alignments.Each sequence is labelled with GenBank Identifier (GI) number andsystematic name of an organism. Secondary structure was predicted byJpred and shown underneath the sequence which was used as a query(designations: H-alpha helix, E-beta strand). CONSENSUS was calculatedfor each alignment column by scaling the sum-of-pairs score within thecolumn between those of a homogeneous column (the same residue in allaligned sequences) and a random column with homogeneity cutoff 0.8.Active site motifs of HEPN domain are shown below alignment. FIG.13A-1-A-2 discloses SEQ ID NOS 385-503, respectively, in order ofappearance. FIG. 13B discloses SEQ ID NOS 504-547, respectively, inorder of appearance. FIGS. 13D-1-H-2 disclose SEQ ID NOS 548-567,respectively, in order of appearance. FIG. 13I-1-13I-4 discloses SEQ IDNOS 568, 569 & 1786, and 570-572, respectively, in order of appearance.FIGS. 13J-1-N-4 disclose SEQ ID NOS 573-591, respectively, in order ofappearance.

FIG. 14 depicts C2c3 neighborhoods, i.e., genomic architecture of theC2c3 CRISPR-Cas loci. The number of repeats in CRISPR arrays isindicated. For each genomic contig, Genbank numeric ID and thecoordinates of the locus are indicated.

FIG. 15 depicts C2c2 neighborhoods, i.e., genomic architecture of theC2c2 CRISPR-Cas loci. The number of repeats in CRISPR arrays isindicated. For each genomic contig, Genbank numeric ID and thecoordinates of the locus are indicated.

FIG. 16-1-16-8 depicts HEPN RxxxxH motif in C2c2 family. FIG. 16-1-16-8discloses SEQ ID NOS 592-1195, respectively, in order of appearance.

FIG. 17 depicts C2C1: 1. Alicyclobacillus acidoterrestris ATCC 49025.FIG. 17 discloses SEQ ID NOS 1196-1199, respectively, in order ofappearance.

FIG. 18 depicts C2C1: 4. Desulfonatronum thiodismutans strain MLF-1.FIG. 18 discloses SEQ ID NOS 1200-1203, respectively, in order ofappearance.

FIG. 19 depicts C2C1: 5. Opitutaceae bacterium TAV5. FIG. 19 disclosesSEQ ID NOS 1204-1207, respectively, in order of appearance.

FIG. 20 depicts C2C1: 7. Bacillus thermoamylovorans strain B4166. FIG.20 discloses SEQ ID NOS 1208-1211, respectively, in order of appearance.

FIG. 21 depicts C2C1: 9. Bacillus sp. NSP2.1. FIG. 21 discloses SEQ IDNOS 1212-1215, respectively, in order of appearance.

FIG. 22 depicts C2C2: 1. Lachnospiraceae bacterium MA2020. FIG. 22discloses SEQ ID NOS 1216-1219, respectively, in order of appearance.

FIG. 23-1-23-2 depicts C2C2: 2. Lachnospiraceae bacterium NK4A179. FIG.23-1-23-2 discloses SEQ ID NOS 1220-1226, respectively, in order ofappearance.

FIG. 24 depicts C2C2: 3. [Clostridium] aminophilum DSM 10710. FIG. 24discloses SEQ ID NOS 1227-1230, respectively, in order of appearance.

FIG. 25 depicts C2C2: 4. Lachnospiraceae bacterium NK4A144. FIG. 25discloses SEQ ID NOS 1231-1232, respectively, in order of appearance.

FIG. 26 depicts C2C2: 5. Carnobacterium gallinarum DSM 4847. FIG. 26discloses SEQ ID NOS 1233-1236, respectively, in order of appearance.

FIG. 27-1-27-2 depicts C2C2: 6. Carnobacterium gallinarum DSM 4847. FIG.27-1-27-2 discloses SEQ ID NOS 1237-1243, respectively, in order ofappearance.

FIG. 28 depicts C2C2: 7. Paludibacter propionicigenes WB4. FIG. 28discloses SEQ ID NO: 1244.

FIG. 29 depicts C2C2: 8. Listeria seeligeri serovar ½b. FIG. 29discloses SEQ ID NOS 1245-1248, respectively, in order of appearance.

FIG. 30 depicts C2C2: 9. Listeria weihenstephanensis FSL R9-0317. FIG.30 discloses SEQ ID NO: 1249.

FIG. 31-1-31-2 depicts C2C2: 10. Listeria bacterium FSL M6-0635. FIG.31-1-31-2 discloses SEQ ID NOS 1250-1253, respectively, in order ofappearance.

FIG. 32 depicts C2C2: 11. Leptotrichia wadei F0279. FIG. 32 disclosesSEQ ID NO: 1254.

FIG. 33-1-33-2 depicts C2C2: 12. Leptotrichia wadei F0279. FIG.33-1-33-2 discloses SEQ ID NOS 1255-1261, respectively, in order ofappearance.

FIG. 34 depicts C2C2: 14. Leptotrichia shahii DSM 19757. FIG. 34discloses SEQ ID NOS 1262-1265, respectively, in order of appearance.

FIG. 35 depicts C2C2: 15. Rhodobacter capsulatus SB 1003. FIG. 35discloses SEQ ID NOS 1266-1267, respectively, in order of appearance.

FIG. 36 depicts C2C2: 16. Rhodobacter capsulatus R121. FIG. 36 disclosesSEQ ID NOS 1268-1269, respectively, in order of appearance.

FIG. 37 depicts C2C2: 17. Rhodobacter capsulatus DE442. FIG. 37discloses SEQ ID NOS 1270-1271, respectively, in order of appearance.

FIG. 38 depicts a tree of DRs.

FIG. 39 depicts a tree of C2C2s.

FIG. 40A-40D shows the Table listing 63 large protein-coding genesidentified using the computational pipeline disclosed herein in thevicinity of cas1 genes. Representatives of the new subtypes disclosedherein (V-B, V-C, VI) are colored. Protein sequences forAUXO014641567.1, AUXO011689375.1, AUXO011689375.1, AUXO011277409.1,AUXO014986615.1 coding representatives of Type V-B and Type IV were notanalyzed, since species affiliation cannot be assigned to thesesequences.

FIG. 41A-41M-2 shows the Table presenting the analysis of Type V-B (C2c1protein-encoding) loci. * cas1cas4—gene containing cas4 and cas1domains; CRISPR—CRISPR repeat; SOS—SOS response gene; unk—hypotheticalprotein; >—direction of gene coding sequence; [D]—degenerate repeat(defined where it was possible); [T]—tracrRNA. FIG. 41C-J shows CRISPRarrays analysis of Type V-B (C2c1 protein-encoding) loci as disclosedherein (CRISPR section is basic output of pilercr (see pilercr site fordescription of output: drive5.com/pilercr/); repeat folding was donewith mfold (see mfold site for description of output: mfold.rnaalbany.edu/?q=mfold/DNA-Folding-Form); repeat folding and CRISPRS arrayare placed after detailed description of each case; for CRISPR locationsee link in the Table in FIG. 41A-B). FIG. 41K shows CRISPRmapclassification of CRISPR repeats of Type V-B (C2c1 protein-encoding)loci as disclosed herein using CRISPRmap (seerna.informatik.uni-freiburg.de/CRISPRmap/Input.jsp for details). FIG.41L shows degenerate repeats of Type V-B (C2c1 protein-encoding) loci asdisclosed herein found using CRISPRs finder (crispr.u-psud.fr/Server/).Normal repeat column contains normal repeat, spacer—the last spacer,downstream—downstream region starting from degenerate repeat (250 bp);array number corresponds to the number of CRISPR array in the respectivelocus (see the Table in FIG. 41A-B); region highlighted in yellow has aperfect match between normal repeat and degenerate repeat (other part ofdegenerate repeat does not match). FIG. 41M-1-41M-2 shows predictedstructures of tracrRNAs base-paired with the repeats. TracrRNA forAlicyclobacillus acidoterrestris was identified using RNAseq. For theremaining loci, putative tracrRNAs were identified based on presence ofan anti-direct repeat (DR) sequence. Anti-DRs were identified usingGeneious (www.geneious.com) by searching for sequences within eachrespective CRISPR locus that are highly homologous to DR. The 5′ and 3′ends of each putative tracrRNA was determined though computationalprediction of bacterial transcription start and termination sites usingBPROM (www.softberry.com) and ARNOLD(ma.ig-mors.u-psud.fr/toolbox/arnold/) respectively. Co-foldingpredictions were generated using Geneious. 5′ ends are colored blue and3′ ends are colored orange. FIG. 41C discloses SEQ ID NOS 1272-1311,respectively, in order of appearance. FIG. 41D discloses SEQ ID NOS1312-1319, respectively, in order of appearance. FIG. 41E discloses SEQID NOS 1320-1326, respectively, in order of appearance. FIG. 41Fdiscloses SEQ ID NOS 1327-1367, respectively, in order of appearance.FIG. 41G discloses SEQ ID NOS 1368-1406, respectively, in order ofappearance. FIG. 41H discloses SEQ ID NOS 1407-1424, respectively, inorder of appearance. FIG. 41I discloses SEQ ID NOS 1425-1460,respectively, in order of appearance. FIG. 41J discloses SEQ ID NOS1461-1471, respectively, in order of appearance. FIG. 41K discloses SEQID NOS 1472-1489, respectively, in order of appearance. FIG. 41Ldiscloses the “Repeat” sequences as SEQ ID NOS 1490-1499, the “Spacer”sequences as SEQ ID NOS 1500-1509, and the “Downstream” sequences as SEQID NOS 1510-1519, all respectively, in order of appearance. FIG. 41Lalso discloses SEQ ID NO: 1520 below the table. FIG. 41M-1-M-2 disclosesSEQ ID NOS 1521-1528, respectively, in order of appearance.

FIG. 42A-42N-2 shows the Table presenting the analysis of Type VI (C2c2protein-encoding) loci. * CRISPR—CRISPR repeat; unk—hypotheticalprotein; >—direction of gene coding sequence; [D]—degenerate repeat(defined where it was possible); [T]-tracrRNA. FIG. 42C-1-I-3 showsCRISPR arrays analysis of Type VI (C2c2 protein-encoding) loci asdisclosed herein (CRISPR section is basic output of pilercr (see pilercrsite for description of output: drive5.com/pilercr/); repeat folding wasdone with mfold (see mfold site for description of output: mfold.rnaalbany.edu/?q=mfold/DNA-Folding-Form); repeat folding and CRISPRS arrayare placed after detailed description of each case; for CRISPR locationsee link in the Table in FIG. 42A-B). FIG. 42J-1-J-2 shows CRISPRmapclassification of CRISPR repeats of Type VI (C2c2 protein-encoding) locias disclosed herein using CRISPRmap (seerna.informatik.uni-freiburg.de/CRISPRmap/Input.jsp for details). FIG.42K-1-L shows degenerate repeats of Type VI (C2c2 protein-encoding) locias disclosed herein found using CRISPRs finder(crispr.u-psud.fr/Server/). Normal repeat column contains normal repeat,spacer—the last spacer, downstream—downstream region starting fromdegenerate repeat (250 bp); array number corresponds to the number ofCRISPR array in the respective locus (see the Table in FIG. 42A-B);region highlighted in yellow has a perfect match between normal repeatand degenerate repeat (other part of degenerate repeat does not match).FIG. 42M-N-2 shows predicted structures of tracrRNAs base-paired withthe repeats. Putative tracrRNAs were identified based on presence of ananti-direct repeat (DR) sequence. Anti-DRs were identified usingGeneious (www.geneious.com) by searching for sequences within eachrespective CRISPR locus that are highly homologous to DR. The 5′ and 3′ends of each putative tracrRNA was determined though computationalprediction of bacterial transcription start and termination sites usingBPROM (www.softberry.com) and ARNOLD(ma.ig-mors.u-psud.fr/toolbox/arnold/) respectively. Co-foldingpredictions were generated using Geneious. 5′ ends are colored blue and3′ ends are colored orange. FIG. 42C-1-C-2 discloses SEQ ID NOS1529-1557, respectively, in order of appearance. FIG. 42D-1-D-2discloses SEQ ID NOS 1558-1583, respectively, in order of appearance.FIG. 42E-1-E-2 discloses SEQ ID NOS 1584-1623, respectively, in order ofappearance. FIG. 42F-1-F-2 discloses SEQ ID NOS 1624-1645, respectively,in order of appearance. FIG. 42G-1-G-2 discloses SEQ ID NOS 1646-1660,respectively, in order of appearance. FIG. 42H-1-H-2 discloses SEQ IDNOS 1661-1678, respectively, in order of appearance. FIG. 42I-1-I-3discloses SEQ ID NOS 1679-1689, respectively, in order of appearance.FIG. 42J-1-J-2 discloses SEQ ID NOS 1690-1719, respectively, in order ofappearance. FIGS. 42K-1-L disclose “Normal Repeat” sequences as SEQ IDNOS 1720-1735, “Spacer” sequences as SEQ ID NOS 1736-1751, and“Downstream” sequences as 1752-1767, all respectively, in order ofappearance. FIG. 42M discloses SEQ ID NOS 1768-1771, respectively, inorder of appearance. FIG. 42N-1-N-2 discloses SEQ ID NOS 1772-1775,respectively, in order of appearance.

FIG. 43A-43F shows the Table presenting the analysis of Type V-C(C2c3protein-encoding) loci. * CRISPR—CRISPR repeat; unk—hypotheticalprotein; >—direction of gene coding sequence; [D]—degenerate repeat(defined where it was possible). FIG. 43C-D-2 shows CRISPR arraysanalysis of Type V-C (C2c3 protein-encoding) loci as disclosed herein(CRISPR section is basic output of CRISPRfinder (see for description:crispr.u-psud.fr/Server/); repeat folding was done with mfold (see mfoldsite for description of output:mfold.rna.albany.edu/?q=mfold/DNA-Folding-Form); repeat folding andCRISPRS array are placed after detailed description of each case; forCRISPR location see link in the Table in FIG. 43A-B). Statisticallysignificant spacer's blast hits in prokaryotes or their viruses areshown. FIG. 43E shows CRISPRmap classification of CRISPR repeats of TypeV-C(C2c3 protein-encoding) loci as disclosed herein using CRISPRmap (seerna.informatik.uni-freiburg.de/CRISPRmap/Input.jsp for details). FIG.43F shows degenerate repeats of Type V-C (C2c3 protein-encoding) loci asdisclosed herein found using CRISPRs finder (crispr.u-psud.fr/Server/).Normal repeat column contains normal repeat, spacer—the last spacer,downstream—downstream region starting from degenerate repeat (250 bp);array number corresponds to the number of CRISPR array in the respectivelocus (see the Table in FIG. 43A-B); region highlighted in yellow has aperfect match between normal repeat and degenerate repeat (other part ofdegenerate repeat does not match). FIG. 43C discloses SEQ ID NOS1776-1785 and 83-104, respectively, in order of appearance. FIG.43D-1-D-2 discloses SEQ ID NOS 105-125, respectively, in order ofappearance. FIG. 43E discloses SEQ ID NOS 126-131, respectively, inorder of appearance. FIG. 43F discloses SEQ ID NOS 132-134,respectively, in order of appearance.

FIG. 44A-44E-2 provides complete list of CRISPR-Cas loci in the genomeswhere C2c1 or C2c2 proteins were found. Genes for C2c1 and C2c2 proteinsare highlighted in yellow. FIGS. 44A-E-2 disclose SEQ ID NOS 135-183,respectively, in order of appearance.

FIG. 45A-45C shows alignment of Listeria loci encoding putative Type VICRISPR-Cas system. The aligned syntenic region corresponds to Listeriaweihenstephanensis FSL R9-0317 contig AODJ01000004.1, coordinates42281-46274 and Listeria newyorkensis strain FSL M6-0635 contigJNFB01000012.1, coordinates 169489-173541. Color coding: C2c2 gene ishighlighted by blue CRISPR repeats—red, degenerated repeat—magenta,spacers—bold. FIGS. 45A-C disclose SEQ ID NOS 184-185, respectively, inorder of appearance.

FIG. 46A-46D shows functional validation of the Alicyclobacillusacidoterrestris C2c1 locus. FIG. 46A: RNA-sequencing shows theAlicyclobacillus acidoterrestris C2c1 locus is highly expressed withprocessed crRNAs incorporating a 5′ 14-nt DR and 20-nt spacer. Aputative 79-nt tracrRNA is expressed robustly in the same orientation asthe cas gene cluster.

FIG. 46B: Northern blot of RNAs expressed from endogenous locus (M) anda minimal first-spacer array (S) show processed crRNAs with a 5′ DR andthe presence of a small putative tracrRNA. Arrows indicate the probepositions and their directionality. FIG. 46C: In silico co-folding ofthe crRNA direct repeat and putative tracrRNA shows stable secondarystructure and complementarity between the two RNAs. 5′ bases are coloredblue and 3′ bases are colored orange. FIG. 46D: Heterologous expressionof the Alicyclobacillus acidoterrestris C2c1 locus in pACYC-184transformed into E. coli shows identical results to the expressionobserved in the endogenous strain (FIG. 46A). Processed crRNAs have a 5′14-nt DR and 20-nt spacer and a putative 79-nt tracrRNA is expressedrobustly. FIG. 46A discloses SEQ ID NOS 186-187, respectively, in orderof appearance. FIG. 46C discloses SEQ ID NOS 188-191, respectively, inorder of appearance. FIG. 46D discloses SEQ ID NOS 192-193,respectively, in order of appearance.

FIG. 47A-47C shows identification of the protospacer adjacent motif(PAM) for the Alicyclobacillus acidoterrestris C2c1 enzyme. FIG. 47A:Schematic of the PAM determination screen. FIG. 47B: Depletion from the5′ left PAM library reveals a 5′ TTN PAM. Depletion is measured as thenegative log 2 fold ratio and PAMs above a threshold of 3.5 are used tocalculate the entropy score at each position. FIG. 47C: Validation ofthe Alicyclobacillus acidoterrestris C2c1 PAM by measuring interferencewith eight different PAMs. PAMs matching the TTN motif show depletion asmeasured by cfus.

FIG. 48A-48F shows in vitro characterization of Alicyclobacillusacidoterrestris C2c1 cleavage requirements. FIG. 48A: in vitro cleavageof the EMX1 target with the human cell lysate expressingAlicyclobacillus acidoterrestris C2c1 shows that in vitro targeting ofAlicyclobacillus acidoterrestris C2c1 is robust and depends on tracrRNA.Non-targeting crRNA (crRNA 2) fails to cleave the EMX1 target, whereascrRNA 1 targeting EMX1 enabled strong cleavage in the presence of Mg++and weak cleavage in the absence of Mg++. FIG. 48B: in vitro cleavage ofthe EMX1 target in the presence of a range of tracrRNA lengthsidentifies the 78 nt species as the minimal tracrRNA form, withincreased cleavage efficiency for the 91nt form.

FIG. 48C: Analysis of the temperature dependency of the in vitrocleavage of the EMX1 target shows that the optimal temperature range ofrobust AacC2c1 cleavage is between 40° C. and 55° C. FIG. 48D: in vitrovalidation of the AacC2c1 PAM requirements with four different PAMs. ThePAMs matching the TTN motif are efficiently cleaved. FIG. 48E: in silicofolding of the chimeric AacC2c1 sgRNA exhibits a stable structure withdirect repeat:anti-direct pairing between segments derived from thetracrRNA (red) and the crRNA (black). FIG. 48F: Comparison of the invitro target cleavage in the presence of crRNA-tracrRNA AacC2c1 andsgRNA identifies comparable cleavage efficiencies. FIG. 48E disclosesSEQ ID NO: 194.

FIG. 49A-49C shows functional validation of the Bacillusthermoamylovorans C2c1 locus. FIG. 49A: Heterologous expression of theBacillus thermoamylovorans C2c1 locus in E. coli. The putative tracrRNAis significantly expressed and is processed to 91 nt. Processed crRNAsare also present with a 5′ 14 nt DR and 19nt spacer. FIG. 49B: In silicoco-folding of the crRNA direct repeat and putative tracrRNA shows stablesecondary structure and complementarity between the two RNAs. 5′ basesare colored blue and 3′ bases are colored orange. FIG. 49C: Depletionfrom the 5′ left PAM library reveals a 5′ ATTN PAM. Depletion ismeasured as the negative log₂ fold ratio and PAMs above a threshold of3.5 are used to calculate the entropy score at each position. FIG. 49Adiscloses SEQ ID NOS 195-196, respectively, in order of appearance. FIG.49B discloses SEQ ID NOS 197-198, respectively, in order of appearance.

FIG. 50 shows all reads at the Bacillus sp. C2C1 locus.

FIG. 51 shows filtered reads between 0 and 55 bp at the Bacillus sp.C2C1 locus.

FIG. 52 shows the co-folding of the DR sequence and the tracr RNAcorresponding to the B. sp. C2C1 locus. Figure discloses SEQ ID NOS199-201, respectively, in order of appearance.

FIG. 53 shows evolutionary scenario for the CRISPR-Cas systems. The Cas8protein is hypothesized to have evolved by inactivation of Cas10 (shownby the white X) which was accompanied by a major acceleration ofevolution. Abbreviations: TR, terminal repeats; TS, terminal sequences;HD, HD family endonuclease; HNH, HNH family endonuclease; RuvC, RuvCfamily endonuclease; HEPN, putative endoribonuclease of HEPNsuperfamily. Genes and protein regions shown in gray denote sequencesthat were encoded in the respective mobile elements but were eliminatedin the course of evolution of CRISPR-Cas systems.

The figures herein are for illustrative purposes only and are notnecessarily drawn to scale.

DETAILED DESCRIPTION OF THE INVENTION

Before the present methods of the invention are described, it is to beunderstood that this invention is not limited to particular methods,components, products or combinations described, as such methods,components, products and combinations may, of course, vary. It is alsoto be understood that the terminology used herein is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

In general, the CRISPR-Cas or CRISPR system is as used in the foregoingdocuments, such as WO 2014/093622 (PCT/JS2013/074667) and referscollectively to transcripts and other elements involved in theexpression of or directing the activity of CRISPR-associated (“Cas”)genes, including sequences encoding a Cas gene, a tracr(trans-activating CRISPR) sequence (e.g. tracrRNA or an active partialtracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and atracrRNA-processed partial direct repeat in the context of an endogenousCRISPR system), a guide sequence (also referred to as a “spacer” in thecontext of an endogenous CRISPR system), or “RNA(s)” as that term isherein used (e.g., RNA(s) to guide Cas, such as Cas9, e.g. CRISPR RNAand transactivating (tracr) RNA or a single guide RNA (sgRNA) (chimericRNA)) or other sequences and transcripts from a CRISPR locus. Ingeneral, a CRISPR system is characterized by elements that promote theformation of a CRISPR complex at the site of a target sequence (alsoreferred to as a protospacer in the context of an endogenous CRISPRsystem). In the context of formation of a CRISPR complex, “targetsequence” refers to a sequence to which a guide sequence is designed tohave complementarity, where hybridization between a target sequence anda guide sequence promotes the formation of a CRISPR complex. A targetsequence may comprise any polynucleotide, such as DNA or RNApolynucleotides. In some embodiments, a target sequence is located inthe nucleus or cytoplasm of a cell. In some embodiments, direct repeatsmay be identified in silico by searching for repetitive motifs thatfulfill any or all of the following criteria: 1. found in a 2 Kb windowof genomic sequence flanking the type II CRISPR locus; 2. span from 20to 50 bp; and 3. interspaced by 20 to 50 bp. In some embodiments, 2 ofthese criteria may be used, for instance 1 and 2, 2 and 3, or 1 and 3.In some embodiments, all 3 criteria may be used.

In embodiments of the invention the terms mature crRNA, single guideRNA, guide sequence and guide RNA, i.e. RNA capable of guiding Cas to atarget genomic locus, are used interchangeably as in foregoing citeddocuments such as WO 2014/093622 (PCT/US2013/074667). In general, aguide sequence is any polynucleotide sequence having sufficientcomplementarity with a target polynucleotide sequence to hybridize withthe target sequence and direct sequence-specific binding of a CRISPRcomplex to the target sequence. In some embodiments, the degree ofcomplementarity between a guide sequence and its corresponding targetsequence, when optimally aligned using a suitable alignment algorithm,is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%,99%, or more. Optimal alignment may be determined with the use of anysuitable algorithm for aligning sequences, non-limiting example of whichinclude the Smith-Waterman algorithm, the Needleman-Wunsch algorithm,algorithms based on the Burrows-Wheeler Transform (e.g. the BurrowsWheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (NovocraftTechnologies; available at www.novocraft.com), ELAND (Illumina, SanDiego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq(available at maq.sourceforge.net). In some embodiments, a guidesequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75,or more nucleotides in length. In some embodiments, a guide sequence isless than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewernucleotides in length. Preferably the guide sequence is 10 30nucleotides long. The ability of a guide sequence to directsequence-specific binding of a CRISPR complex to a target sequence maybe assessed by any suitable assay. For example, the components of aCRISPR system sufficient to form a CRISPR complex, including the guidesequence to be tested, may be provided to a host cell having thecorresponding target sequence, such as by transfection with vectorsencoding the components of the CRISPR sequence, followed by anassessment of preferential cleavage within the target sequence, such asby Surveyor assay as described herein. Similarly, cleavage of a targetpolynucleotide sequence may be evaluated in a test tube by providing thetarget sequence, components of a CRISPR complex, including the guidesequence to be tested and a control guide sequence different from thetest guide sequence, and comparing binding or rate of cleavage at thetarget sequence between the test and control guide sequence reactions.Other assays are possible, and will occur to those skilled in the art.

In a classic CRISPR-Cas systems, the degree of complementarity between aguide sequence and its corresponding target sequence can be about ormore than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or 100%;a guide or RNA or sgRNA can be about or more than about 5, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,35, 40, 45, 50, 75, or more nucleotides in length; or guide or RNA orsgRNA can be less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, orfewer nucleotides in length; and advantageously tracr RNA is 30 or 50nucleotides in length. However, an aspect of the invention is to reduceoff-target interactions, e.g., reduce the guide interacting with atarget sequence having low complementarity. Indeed, in the examples, itis shown that the invention involves mutations that result in theCRISPR-Cas system being able to distinguish between target andoff-target sequences that have greater than 80% to about 95%complementarity, e.g., 83%-84% or 88-89% or 94-95% complementarity (forinstance, distinguishing between a target having 18 nucleotides from anoff-target of 18 nucleotides having 1, 2 or 3 mismatches). Accordingly,in the context of the present invention the degree of complementaritybetween a guide sequence and its corresponding target sequence isgreater than 94.5% or 95% or 95.5% or 96% or 96.5% or 97% or 97.5% or98% or 98.5% or 99% or 99.5% or 99.9%, or 100%. Off target is less than100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97% or96.5% or 96% or 95.5% or 95% or 94.5% or 94% or 93% or 92% or 91% or 90%or 89% or 88% or 87% or 86% or 85% or 84% or 83% or 82% or 81% or 80%complementarity between the sequence and the guide, with it advantageousthat off target is 100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98%or 97.5% or 97% or 96.5% or 96% or 95.5% or 95% or 94.5% complementaritybetween the sequence and the guide.

In particularly preferred embodiments according to the invention, theguide RNA (capable of guiding Cas to a target locus) may comprise (1) aguide sequence capable of hybridizing to a genomic target locus in theeukaryotic cell; (2) a tracr sequence; and (3) a tracr mate sequence.All (1) to (3) may reside in a single RNA, i.e. an sgRNA (arranged in a5′ to 3′ orientation), or the tracr RNA may be a different RNA than theRNA containing the guide and tracr sequence. The tracr hybridizes to thetracr mate sequence and directs the CRISPR/Cas complex to the targetsequence.

Aspects of the invention relate to the identification and engineering ofnovel effector proteins associated with Class 2 CRISPR-Cas systems. In apreferred embodiment, the effector protein comprises a single-subuniteffector module. In a further embodiment the effector protein isfunctional in prokaryotic or eukaryotic cells for in vitro, in vivo orex vivo applications. An aspect of the invention encompassescomputational methods and algorithms to predict new Class 2 CRISPR-Cassystems and identify the components therein.

In one embodiment, a computational method of identifying novel Class 2CRISPR-Cas loci comprises the following steps: detecting all contigsencoding the Cas1 protein; identifying all predicted protein codinggenes within 20 kB of the cas1 gene; comparing the identified genes withCas protein-specific profiles and predicting CRISPR arrays; selectingunclassified candidate CRISPR-Cas loci containing proteins larger than500 amino acids (>500 aa); analyzing selected candidates using PSI-BLASTand HHPred, thereby isolating and identifying novel Class 2 CRISPR-Casloci. In addition to the above mentioned steps, additional analysis ofthe candidates may be conducted by searching metagenomics databases foradditional homologs.

In one aspect the detecting all contigs encoding the Cas1 protein isperformed by GenemarkS which a gene prediction program as furtherdescribed in “GeneMarkS: a self-training method for prediction of genestarts in microbial genomes. Implications for finding sequence motifs inregulatory regions.” John Besemer, Alexandre Lomsadze and MarkBorodovsky, Nucleic Acids Research (2001) 29, pp 2607-2618, hereinincorporated by reference.

In one aspect the identifying all predicted protein coding genes iscarried out by comparing the identified genes with Cas protein-specificprofiles and annotating them according to NCBI Conserved Domain Database(CDD) which is a protein annotation resource that consists of acollection of well-annotated multiple sequence alignment models forancient domains and full-length proteins. These are available asposition-specific score matrices (PSSMs) for fast identification ofconserved domains in protein sequences via RPS-BLAST. CDD contentincludes NCBI-curated domains, which use 3D-structure information toexplicitly define domain boundaries and provide insights intosequence/structure/function relationships, as well as domain modelsimported from a number of external source databases (Pfam, SMART, COG,PRK, TIGRFAM). In a further aspect, CRISPR arrays were predicted using aPILER-CR program which is a public domain software for finding CRISPRrepeats as described in “PILER-CR: fast and accurate identification ofCRISPR repeats”, Edgar, R. C., BMC Bioinformatics, January 20; 8:18(2007), herein incorporated by reference.

In a further aspect, the case by case analysis is performed usingPSI-BLAST (Position-Specific Iterative Basic Local Alignment SearchTool). PSI-BLAST derives a position-specific scoring matrix (PSSM) orprofile from the multiple sequence alignment of sequences detected abovea given score threshold using protein-protein BLAST. This PSSM is usedto further search the database for new matches, and is updated forsubsequent iterations with these newly detected sequences. Thus,PSI-BLAST provides a means of detecting distant relationships betweenproteins.

In another aspect, the case by case analysis is performed using HHpred,a method for sequence database searching and structure prediction thatis as easy to use as BLAST or PSI-BLAST and that is at the same timemuch more sensitive in finding remote homologs. In fact, HHpred'ssensitivity is competitive with the most powerful servers for structureprediction currently available. HHpred is the first server that is basedon the pairwise comparison of profile hidden Markov models (HMMs).Whereas most conventional sequence search methods search sequencedatabases such as UniProt or the NR, HHpred searches alignmentdatabases, like Pfam or SMART. This greatly simplifies the list of hitsto a number of sequence families instead of a clutter of singlesequences. All major publicly available profile and alignment databasesare available through HHpred. HHpred accepts a single query sequence ora multiple alignment as input. Within only a few minutes it returns thesearch results in an easy-to-read format similar to that of PSI-BLAST.Search options include local or global alignment and scoring secondarystructure similarity. HHpred can produce pairwise query-templatesequence alignments, merged query-template multiple alignments (e.g. fortransitive searches), as well as 3D structural models calculated by theMODELLER software from HHpred alignments.

The term “nucleic acid-targeting system”, wherein nucleic acid is DNA orRNA, and in some aspects may also refer to DNA-RNA hybirds orderivatives thereof, refers collectively to transcripts and otherelements involved in the expression of or directing the activity of DNAor RNA-targeting CRISPR-associated (“Cas”) genes, which may includesequences encoding a DNA or RNA-targeting Cas protein and a DNA orRNA-targeting guide RNA comprising a CRISPR RNA (crRNA) sequence and (inCRISPR-Cas9 system but not all systems) a trans-activating CRISPR-Cassystem RNA (tracrRNA) sequence, or other sequences and transcripts froma DNA or RNA-targeting CRISPR locus. In general, a RNA-targeting systemis characterized by elements that promote the formation of aRNA-targeting complex at the site of a target RNA sequence. In thecontext of formation of a DNA or RNA-targeting complex, “targetsequence” refers to a DNA or RNA sequence to which a DNA orRNA-targeting guide RNA is designed to have complementarity, wherehybridization between a target sequence and a RNA-targeting guide RNApromotes the formation of a RNA-targeting complex. In some embodiments,a target sequence is located in the nucleus or cytoplasm of a cell.

In an aspect of the invention, novel DNA targeting systems also referredto as DNA-targeting CRISPR-Cas or the CRISPR-Cas DNA-targeting system ofthe present application are based on identified Type V (e.g. subtype V-Aand subtype V-B) Cas proteins which do not require the generation ofcustomized proteins to target specific DNA sequences but rather a singleeffector protein or enzyme can be programmed by a RNA molecule torecognize a specific DNA target, in other words the enzyme can berecruited to a specific DNA target using said RNA molecule. Aspects ofthe invention particularly relate to DNA targeting RNA-guided C2c1 orC2c3 CRISPR systems.

In an aspect of the invention, novel RNA targeting systems also referredto as RNA- or RNA-targeting CRISPR-Cas or the CRISPR-Cas systemRNA-targeting system of the present application are based on identifiedType VI Cas proteins which do not require the generation of customizedproteins to target specific RNA sequences but rather a single enzyme canbe programmed by a RNA molecule to recognize a specific RNA target, inother words the enzyme can be recruited to a specific RNA target usingsaid RNA molecule.

As used herein, a Cas protein or a CRISPR enzyme refers to any of theproteins presented in the new classification of CRISPR-Cas systems. Inan advantageous embodiment, the present invention encompasses effectorproteins identified in a Type V CRISPR-Cas loci, e.g. a Cpf1-encodingloci denoted as subtype V-A. Presently, the subtype V-A loci encompassescas1, cas2, a distinct gene denoted cpf1 and a CRISPR array. Cpf1(CRISPR-associated protein Cpf1, subtype PREFRAN) is a large protein(about 1300 amino acids) that contains a RuvC-like nuclease domainhomologous to the corresponding domain of Cas9 along with a counterpartto the characteristic arginine-rich cluster of Cas9. However, Cpf1 lacksthe HNH nuclease domain that is present in all Cas9 proteins, and theRuvC-like domain is contiguous in the Cpf1 sequence, in contrast to Cas9where it contains long inserts including the HNH domain. Accordingly, inparticular embodiments, the CRISPR-Cas enzyme comprises only a RuvC-likenuclease domain.

In an advantageous embodiment, the present invention encompassescompositions and systems comprising effector proteins identified in aC2c1 loci denoted as subtype V-B. Herein, C2c1 refers to Class 2candidate 1. All C2c1 loci encode a Cas1-Cas4 fusion, Cas2, and thelarge protein Applicants denote as C2c1p, and typically, are adjacent toa CRISPR array.

In an advantageous embodiment, the present invention encompasseseffector proteins identified in a Type VI CRISPR-Cas loci, e.g. the C2c2loci. Herein, C2c2 refers to Class 2 candidate 2. The C2c2 lociencompass cas1 and cas2 genes along with the large protein Applicantsdenote as C2c2p, and a CRISPR array; however, unlike C2c1p, C2c2p isoften encoded next to a CRISPR array but not cas1-cas2.

In an advantageous embodiment, the present invention encompassescompositions and systems comprising effector proteins identified in aC2c3 loci denoted as subtype V-C. Herein, C2c3 refers to Class 2candidate 3. All C2c3 loci encode a Cas1-Cas4 fusion, Cas2, and thelarge protein Applicants denote as C2c3p.

Aspects of the invention also encompass methods and uses of thecompositions and systems described herein in genome engineering, e.g.for altering or manipulating the expression of one or more genes or theone or more gene products, in prokaryotic or eukaryotic cells, in vitro,in vivo or ex vivo.

The nucleic acids-targeting systems, the vector systems, the vectors andthe compositions described herein may be used in various nucleicacids-targeting applications, altering or modifying synthesis of a geneproduct, such as a protein, nucleic acids cleavage, nucleic acidsediting, nucleic acids splicing; trafficking of target nucleic acids,tracing of target nucleic acids, isolation of target nucleic acids,visualization of target nucleic acids, etc.

Aspects of the invention also encompass methods and uses of thecompositions and systems described herein in genome engineering, e.g.for altering or manipulating the expression of one or more genes or theone or more gene products, in prokaryotic or eukaryotic cells, in vitro,in vivo or ex vivo.

The methods according to the invention as described herein comprehendinducing one or more mutations in a eukaryotic cell (in vitro, i.e. inan isolated eukaryotic cell) as herein discussed comprising deliveringto cell a vector as herein discussed. The mutation(s) can include theintroduction, deletion, or substitution of one or more nucleotides ateach target sequence of cell(s) via the guide(s) RNA(s) or sgRNA(s). Themutations can include the introduction, deletion, or substitution of1-75 nucleotides at each target sequence of said cell(s) via theguide(s) RNA(s) or sgRNA(s). The mutations can include the introduction,deletion, or substitution of 1, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75nucleotides at each target sequence of said cell(s) via the guide(s)RNA(s) or sgRNA(s). The mutations can include the introduction,deletion, or substitution of 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75nucleotides at each target sequence of said cell(s) via the guide(s)RNA(s) or sgRNA(s). The mutations include the introduction, deletion, orsubstitution of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides at eachtarget sequence of said cell(s) via the guide(s) RNA(s) or sgRNA(s). Themutations can include the introduction, deletion, or substitution of 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75nucleotides at each target sequence of said cell(s) via the guide(s)RNA(s) or sgRNA(s). The mutations can include the introduction,deletion, or substitution of 40, 45, 50, 75, 100, 200, 300, 400 or 500nucleotides at each target sequence of said cell(s) via the guide(s)RNA(s) or sgRNA(s).

For minimization of toxicity and off-target effect, it will be importantto control the concentration of Cas mRNA and guide RNA delivered.Optimal concentrations of Cas mRNA and guide RNA can be determined bytesting different concentrations in a cellular or non-human eukaryoteanimal model and using deep sequencing the analyze the extent ofmodification at potential off-target genomic loci. Alternatively, tominimize the level of toxicity and off-target effect, Cas nickase mRNA(for example S. pyogenes Cas9 with the D10A mutation) can be deliveredwith a pair of guide RNAs targeting a site of interest. Guide sequencesand strategies to minimize toxicity and off-target effects can be as inWO 2014/093622 (PCT/US2013/074667); or, via mutation as herein.

Typically, in the context of an endogenous CRISPR system, formation of aCRISPR complex (comprising a guide sequence hybridized to a targetsequence and complexed with one or more Cas proteins) results incleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence.Without wishing to be bound by theory, the tracr sequence, which maycomprise or consist of all or a portion of a wild-type tracr sequence(e.g. about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, ormore nucleotides of a wild-type tracr sequence), may also form part of aCRISPR complex, such as by hybridization along at least a portion of thetracr sequence to all or a portion of a tracr mate sequence that isoperably linked to the guide sequence.

The nucleic acid molecule encoding a Cas is advantageously codonoptimized Cas. An example of a codon optimized sequence, is in thisinstance a sequence optimized for expression in a eukaryote, e.g.,humans (i.e. being optimized for expression in humans), or for anothereukaryote, animal or mammal as herein discussed; see, e.g., SaCas9 humancodon optimized sequence in WO 2014/093622 (PCT/US2013/074667). Whilstthis is preferred, it will be appreciated that other examples arepossible and codon optimization for a host species other than human, orfor codon optimization for specific organs is known. In someembodiments, an enzyme coding sequence encoding a Cas is codon optimizedfor expression in particular cells, such as eukaryotic cells. Theeukaryotic cells may be those of or derived from a particular organism,such as a mammal, including but not limited to human, or non-humaneukaryote or animal or mammal as herein discussed, e.g., mouse, rat,rabbit, dog, livestock, or non-human mammal or primate. In someembodiments, processes for modifying the germ line genetic identity ofhuman beings and/or processes for modifying the genetic identity ofanimals which are likely to cause them suffering without any substantialmedical benefit to man or animal, and also animals resulting from suchprocesses, may be excluded. In general, codon optimization refers to aprocess of modifying a nucleic acid sequence for enhanced expression inthe host cells of interest by replacing at least one codon (e.g. aboutor more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) ofthe native sequence with codons that are more frequently or mostfrequently used in the genes of that host cell while maintaining thenative amino acid sequence. Various species exhibit particular bias forcertain codons of a particular amino acid. Codon bias (differences incodon usage between organisms) often correlates with the efficiency oftranslation of messenger RNA (mRNA), which is in turn believed to bedependent on, among other things, the properties of the codons beingtranslated and the availability of particular transfer RNA (tRNA)molecules. The predominance of selected tRNAs in a cell is generally areflection of the codons used most frequently in peptide synthesis.Accordingly, genes can be tailored for optimal gene expression in agiven organism based on codon optimization. Codon usage tables arereadily available, for example, at the “Codon Usage Database” availableat www.kazusa.orjp/codon/ and these tables can be adapted in a number ofways. See Nakamura, Y., et al. “Codon usage tabulated from theinternational DNA sequence databases: status for the year 2000” Nucl.Acids Res. 28:292 (2000). Computer algorithms for codon optimizing aparticular sequence for expression in a particular host cell are alsoavailable, such as Gene Forge (Aptagen; Jacobus, Pa.), are alsoavailable. In some embodiments, one or more codons (e.g. 1, 2, 3, 4, 5,10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a Cascorrespond to the most frequently used codon for a particular aminoacid.

In certain embodiments, the methods as described herein may compriseproviding a Cas transgenic cell in which one or more nucleic acidsencoding one or more guide RNAs are provided or introduced operablyconnected in the cell with a regulatory element comprising a promoter ofone or more gene of interest. As used herein, the term “Cas transgeniccell” refers to a cell, such as a eukaryotic cell, in which a Cas genehas been genomically integrated. The nature, type, or origin of the cellare not particularly limiting according to the present invention. Alsothe way how the Cas transgene is introduced in the cell is may vary andcan be any method as is known in the art. In certain embodiments, theCas transgenic cell is obtained by introducing the Cas transgene in anisolated cell. In certain other embodiments, the Cas transgenic cell isobtained by isolating cells from a Cas transgenic organism. By means ofexample, and without limitation, the Cas transgenic cell as referred toherein may be derived from a Cas transgenic eukaryote, such as a Casknock-in eukaryote. Reference is made to WO 2014/093622(PCT/US13/74667), incorporated herein by reference. Methods of U.S. Pat.No. 8,771,985 assigned to Sangamo BioSciences, Inc. and Sigma-AldrichCo. LLC and US Patent Publication No. 20110265198 assigned to SangamoBioSciences, Inc. directed to targeting the Rosa locus may be modifiedto utilize the CRISPR Cas system of the present invention. Methods of USPatent Publication No. 20130236946 assigned to Cellectis directed totargeting the Rosa locus may also be modified to utilize the CRISPR Cassystem of the present invention. By means of further example referenceis made to Platt et. al. (Cell; 159(2):440-455 (2014)), describing aCas9 knock-in mouse, which is incorporated herein by reference. The Castransgene can further comprise a Lox-Stop-polyA-Lox (LSL) cassettethereby rendering Cas expression inducible by Cre recombinase.Alternatively, the Cas transgenic cell may be obtained by introducingthe Cas transgene in an isolated cell. Delivery systems for transgenesare well known in the art. By means of example, the Cas transgene may bedelivered in for instance eukaryotic cell by means of vector (e.g., AAV,adenovirus, lentivirus) and/or particle and/or nanoparticle delivery, asalso described herein elsewhere.

It will be understood by the skilled person that the cell, such as theCas transgenic cell, as referred to herein may comprise further genomicalterations besides having an integrated Cas gene or the mutationsarising from the sequence specific action of Cas when complexed with RNAcapable of guiding Cas to a target locus, such as for instance one ormore oncogenic mutations, as for instance and without limitationdescribed in Platt et al. (2014), Chen et al., (2014) or Kumar et al.(2009).

In some embodiments, the Cas sequence is fused to one or more nuclearlocalization sequences (NLSs), such as about or more than about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, the Cascomprises about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore NLSs at or near the amino-terminus, about or more than about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the carboxy-terminus,or a combination of these (e.g. zero or at least one or more NLS at theamino-terminus and zero or at one or more NLS at the carboxy terminus).When more than one NLS is present, each may be selected independently ofthe others, such that a single NLS may be present in more than one copyand/or in combination with one or more other NLSs present in one or morecopies. In a preferred embodiment of the invention, the Cas comprises atmost 6 NLSs. In some embodiments, an NLS is considered near the N- orC-terminus when the nearest amino acid of the NLS is within about 1, 2,3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along thepolypeptide chain from the N- or C-terminus. Non-limiting examples ofNLSs include an NLS sequence derived from: the NLS of the SV40 viruslarge T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 1);the NLS from nucleoplasmin (e.g. the nucleoplasmin bipartite NLS withthe sequence KRPAATKKAGQAKKKK) (SEQ ID NO: 2); the c-myc NLS having theamino acid sequence PAAKRVKLD (SEQ ID NO: 3) or RQRRNELKRSP (SEQ ID NO:4); the hRNPA1 M9 NLS having the sequenceNQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 5); the sequenceRMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 6) of the IBBdomain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 7) andPPKKARED (SEQ ID NO: 8) of the myoma T protein; the sequence PQPKKKPL(SEQ ID NO: 9) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 10)of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 11) and PKQKKRK (SEQID NO: 12) of the influenza virus NS1; the sequence RKLKKKIKKL (SEQ IDNO: 13) of the Hepatitis virus delta antigen; the sequence REKKKFLKRR(SEQ ID NO: 14) of the mouse Mx1 protein; the sequenceKRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 15) of the human poly(ADP-ribose)polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 16) of thesteroid hormone receptors (human) glucocorticoid. In general, the one ormore NLSs are of sufficient strength to drive accumulation of the Cas ina detectable amount in the nucleus of a eukaryotic cell. In general,strength of nuclear localization activity may derive from the number ofNLSs in the Cas, the particular NLS(s) used, or a combination of thesefactors. Detection of accumulation in the nucleus may be performed byany suitable technique. For example, a detectable marker may be fused tothe Cas, such that location within a cell may be visualized, such as incombination with a means for detecting the location of the nucleus (e.g.a stain specific for the nucleus such as DAPI). Cell nuclei may also beisolated from cells, the contents of which may then be analyzed by anysuitable process for detecting protein, such as immunohistochemistry,Western blot, or enzyme activity assay. Accumulation in the nucleus mayalso be determined indirectly, such as by an assay for the effect ofCRISPR complex formation (e.g. assay for DNA cleavage or mutation at thetarget sequence, or assay for altered gene expression activity affectedby CRISPR complex formation and/or Cas enzyme activity), as compared toa control no exposed to the Cas or complex, or exposed to a Cas lackingthe one or more NLSs.

In certain aspects the invention involves vectors, e.g. for deliveringor introducing in a cell Cas and/or RNA capable of guiding Cas to atarget locus (i.e. guide RNA), but also for propagating these components(e.g. in prokaryotic cells). A used herein, a “vector” is a tool thatallows or facilitates the transfer of an entity from one environment toanother. It is a replicon, such as a plasmid, phage, or cosmid, intowhich another DNA segment may be inserted so as to bring about thereplication of the inserted segment. Generally, a vector is capable ofreplication when associated with the proper control elements. Ingeneral, the term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. Vectorsinclude, but are not limited to, nucleic acid molecules that aresingle-stranded, double-stranded, or partially double-stranded; nucleicacid molecules that comprise one or more free ends, no free ends (e.g.circular); nucleic acid molecules that comprise DNA, RNA, or both; andother varieties of polynucleotides known in the art. One type of vectoris a “plasmid,” which refers to a circular double stranded DNA loop intowhich additional DNA segments can be inserted, such as by standardmolecular cloning techniques. Another type of vector is a viral vector,wherein virally-derived DNA or RNA sequences are present in the vectorfor packaging into a virus (e.g. retroviruses, replication defectiveretroviruses, adenoviruses, replication defective adenoviruses, andadeno-associated viruses (AAVs)). Viral vectors also includepolynucleotides carried by a virus for transfection into a host cell.Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (e.g. bacterial vectors having abacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively-linked. Such vectors are referred to herein as “expressionvectors.” Vectors for and that result in expression in a eukaryotic cellcan be referred to herein as “eukaryotic expression vectors.” Commonexpression vectors of utility in recombinant DNA techniques are often inthe form of plasmids.

Recombinant expression vectors can comprise a nucleic acid of theinvention in a form suitable for expression of the nucleic acid in ahost cell, which means that the recombinant expression vectors includeone or more regulatory elements, which may be selected on the basis ofthe host cells to be used for expression, that is operatively-linked tothe nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory element(s)in a manner that allows for expression of the nucleotide sequence (e.g.in an in vitro transcription/translation system or in a host cell whenthe vector is introduced into the host cell). With regards torecombination and cloning methods, mention is made of U.S. patentapplication Ser. No. 10/815,730, published Sep. 2, 2004 as US2004-0171156 A1, the contents of which are herein incorporated byreference in their entirety.

The vector(s) can include the regulatory element(s), e.g., promoter(s).The vector(s) can comprise Cas encoding sequences, and/or a single, butpossibly also can comprise at least 3 or 8 or 16 or 32 or 48 or 50 guideRNA(s) (e.g., sgRNAs) encoding sequences, such as 1-2, 1-3, 1-4 1-5,3-6, 3-7, 3-8, 3-9, 3-10, 3-8, 3-16, 3-30, 3-32, 3-48, 3-50 RNA(s)(e.g., sgRNAs). In a single vector there can be a promoter for each RNA(e.g., sgRNA), advantageously when there are up to about 16 RNA(s)(e.g., sgRNAs); and, when a single vector provides for more than 16RNA(s) (e.g., sgRNAs), one or more promoter(s) can drive expression ofmore than one of the RNA(s) (e.g., sgRNAs), e.g., when there are 32RNA(s) (e.g., sgRNAs), each promoter can drive expression of two RNA(s)(e.g., sgRNAs), and when there are 48 RNA(s) (e.g., sgRNAs), eachpromoter can drive expression of three RNA(s) (e.g., sgRNAs). By simplearithmetic and well established cloning protocols and the teachings inthis disclosure one skilled in the art can readily practice theinvention as to the RNA(s) (e.g., sgRNA(s) for a suitable exemplaryvector such as AAV, and a suitable promoter such as the U6 promoter,e.g., U6-sgRNAs. For example, the packaging limit of AAV is −4.7 kb. Thelength of a single U6-sgRNA (plus restriction sites for cloning) is 361bp. Therefore, the skilled person can readily fit about 12-16, e.g., 13U6-sgRNA cassettes in a single vector. This can be assembled by anysuitable means, such as a golden gate strategy used for TALE assembly(www.genome-engineering.org/taleffectors/). The skilled person can alsouse a tandem guide strategy to increase the number of U6-sgRNAs byapproximately 1.5 times, e.g., to increase from 12-16, e.g., 13 toapproximately 18-24, e.g., about 19 U6-sgRNAs. Therefore, one skilled inthe art can readily reach approximately 18-24, e.g., about 19promoter-RNAs, e.g., U6-sgRNAs in a single vector, e.g., an AAV vector.A further means for increasing the number of promoters and RNAs, e.g.,sgRNA(s) in a vector is to use a single promoter (e.g., U6) to expressan array of RNAs, e.g., sgRNAs separated by cleavable sequences. And aneven further means for increasing the number of promoter-RNAs, e.g.,sgRNAs in a vector, is to express an array of promoter-RNAs, e.g.,sgRNAs separated by cleavable sequences in the intron of a codingsequence or gene; and, in this instance it is advantageous to use apolymerase II promoter, which can have increased expression and enablethe transcription of long RNA in a tissue specific manner. (see, e.g.,nar.oxfordjournals.org/content/34/7/e53.short,www.nature.com/mt/journal/v16/n9/abs/mt2008144a.html). In anadvantageous embodiment, AAV may package U6 tandem sgRNA targeting up toabout 50 genes. Accordingly, from the knowledge in the art and theteachings in this disclosure the skilled person can readily make and usevector(s), e.g., a single vector, expressing multiple RNAs or guides orsgRNAs under the control or operatively or functionally linked to one ormore promoters-especially as to the numbers of RNAs or guides or sgRNAsdiscussed herein, without any undue experimentation.

The guide RNA(s), e.g., sgRNA(s) encoding sequences and/or Cas encodingsequences, can be functionally or operatively linked to regulatoryelement(s) and hence the regulatory element(s) drive expression. Thepromoter(s) can be constitutive promoter(s) and/or conditionalpromoter(s) and/or inducible promoter(s) and/or tissue specificpromoter(s). The promoter can be selected from the group consisting ofRNA polymerases, pol I, pol II, pol III, T7, U6, H1, retroviral Roussarcoma virus (RSV) LTR promoter, the cytomegalovirus (CMV) promoter,the SV40 promoter, the dihydrofolate reductase promoter, the β-actinpromoter, the phosphoglycerol kinase (PGK) promoter, and the EF1αpromoter. An advantageous promoter is the promoter is U6.

In general, the CRISPR-Cas9 system is as used in the foregoingdocuments, such as WO 2014/093622 (PCT/US2013/074667) and referscollectively to transcripts and other elements involved in theexpression of or directing the activity of CRISPR-associated (“Cas”)enzyme, e.g. Cas9, including sequences encoding or delivering a Casenzyme (DNA and/or RNA-targeting) enzyme, a tracr (trans-activatingCRISPR) sequence (e.g., tracrRNA or an active partial tracrRNA), atracr-mate sequence (encompassing a “direct repeat” and atracrRNA-processed partial direct repeat in the context of an endogenousCRISPR system), a guide sequence (also referred to as a “spacer” in thecontext of an endogenous CRISPR system), or “RNA(s)” as that term isherein used (e.g., RNA(s) to guide Cas9, e.g., CRISPR RNA (crRNA) andtrans-activating crRNA (tracrRNA) or a single guide RNA (sgRNA)(chimeric RNA)) or other sequences and transcripts from a CRISPR locus.In general, a CRISPR system is characterized by elements that promotethe formation of a CRISPR complex at the site of a target sequence (alsoreferred to as a protospacer in the context of an endogenous CRISPRsystem). In the context of formation of a CRISPR complex, “targetsequence” refers to a sequence to which a guide sequence is designed totarget, e.g. have complementarity, where hybridization between a targetsequence and a guide sequence promotes the formation of a CRISPRcomplex. The section of the guide sequence through which complementarityto the target sequence is important for cleavage activity is referred toherein as the seed sequence. A target sequence may comprise anypolynucleotide, such as DNA or RNA polynucleotides and is comprisedwithin a target locus of interest. In some embodiments, a targetsequence is located in the nucleus or cytoplasm of a cell. The hereindescribed invention encompasses novel effector proteins of Class 2CRISPR-Cas systems, of which Cas9 is an exemplary effector protein andhence terms used in this application to describe novel effectorproteins, may correlate to the terms used to describe the CRISPR-Cas9system.

The CRISPR-Cas loci has more than 50 gene families and there is nostrictly universal genes. Therefore, no single evolutionary tree isfeasible and a multi-pronged approach is needed to identify newfamilies. So far, there is comprehensive cas gene identification of 395profiles for 93 Cas proteins. Classification includes signature geneprofiles plus signatures of locus architecture. A new classification ofCRISPR-Cas systems is proposed in FIGS. 1A and 1B. Class 1 includesmultisubunit crRNA-effector complexes (Cascade) and Class 2 includesSingle-subunit crRNA-effector complexes (Cas9-like). FIG. 2 provides amolecular organization of CRISPR-Cas. FIG. 3A-3D provides structures ofType I and III effector complexes: common architecture/common ancestrydespite extensive sequence divergence. FIG. 4 shows CRISPR-Cas as a RNArecognition motif (RRM)-centered system. FIG. 5 shows Cas1 phylogenywhere recombination of adaptation and crRNA-effector modules show amajor aspect of CRISPR-Cas evolution. FIG. 6 shows a CRISPR-Cas census,specifically a distribution of CRISPR-Cas types/subtypes among archaeaand bacteria.

The action of the CRISPR-Cas system is usually divided into threestages: (1) adaptation or spacer integration, (2) processing of theprimary transcript of the CRISPR locus (pre-crRNA) and maturation of thecrRNA which includes the spacer and variable regions corresponding to 5′and 3′ fragments of CRISPR repeats, and (3) DNA (or RNA) interference.Two proteins, Cas1 and Cas2, that are present in the great majority ofthe known CRISPR-Cas systems are sufficient for the insertion of spacersinto the CRISPR cassettes. These two proteins form a complex that isrequired for this adaptation process; the endonuclease activity of Cas1is required for spacer integration whereas Cas2 appears to perform anonenzymatic function. The Cas1-Cas2 complex represents the highlyconserved “information processing” module of CRISPR-Cas that appears tobe quasi-autonomous from the rest of the system. (See Annotation andClassification of CRISPR-Cas Systems. Makarova K S, Koonin E V. MethodsMol Biol. 2015; 1311:47-75).

The previously described Class 2 systems, namely Type II and theputative Type V, consisted of only three or four genes in the casoperon, namely the cas1 and cas2 genes comprising the adaptation module(the cas1-cas2 pair of genes are not involved in interference), a singlemultidomain effector protein that is responsible for interference butalso contributes to the pre-crRNA processing and adaptation, and often afourth gene with uncharacterized functions that is dispensable in atleast some Type II systems (and in some cases the fourth gene is cas4(biochemical or in silico evidence shows that Cas4 is a PD−(DE)×Ksuperfamily nuclease with three-cysteine C-terminal cluster; possesses5′-ssDNA exonuclease activity) or csn2, which encodes an inactivatedATPase). In most cases, a CRISPR array and a gene for a distinct RNAspecies known as tracrRNA, a trans-encoded small CRISPR RNA, areadjacent to Class 2 cas operons. The tracrRNA is partially homologous tothe repeats within the respective CRISPR array and is essential for theprocessing of pre-crRNA that is catalyzed by RNAse III, a ubiquitousbacterial enzyme that is not associated with the CRISPR-cas loci.

Cas1 is the most conserved protein that is present in most of theCRISPR-Cas systems and evolves slower than other Cas proteins.Accordingly, Cas1 phylogeny has been used as the guide for CRISPR-Cassystem classification. Biochemical or in silico evidence shows that Cas1is a metal-dependent deoxyribonuclease. Deletion of Cas1 in E. coliresults in increased sensitivity to DNA damage and impaired chromosomalsegregation as described in “A dual function of the CRISPR-Cas system inbacterial antivirus immunity and DNA repair,” Babu M et al. MolMicrobiol 79:484-502 (2011). Biochemical or in silico evidence showsthat Cas 2 is a RNase specific to U-rich regions and is adouble-stranded DNase.

Aspects of the invention relate to the identification and engineering ofnovel effector proteins associated with Class 2 CRISPR-Cas systems. In apreferred embodiment, the effector protein comprises a single-subuniteffector module. In a further embodiment the effector protein isfunctional in prokaryotic or eukaryotic cells for in vitro, in vivo orex vivo applications. An aspect of the invention encompassescomputational methods and algorithms to predict new Class 2 CRISPR-Cassystems and identify the components therein.

In one embodiment, a computational method of identifying novel Class 2CRISPR-Cas loci comprises the following steps: detecting all contigsencoding the Cas1 protein; identifying all predicted protein codinggenes within 20 kB of the cas1 gene, more particularly within the region20 kb from the start of the cas1 gene and 20 kb from the end of the cas1gene; comparing the identified genes with Cas protein-specific profilesand predicting CRISPR arrays; selecting partial and/or unclassifiedcandidate CRISPR-Cas loci containing proteins larger than 500 aminoacids (>500 aa); analyzing selected candidates using PSI-BLAST andHHPred, thereby isolating and identifying novel Class 2 CRISPR-Cas loci.In addition to the above mentioned steps, additional analysis of thecandidates may be conducted by searching metagenomics databases foradditional homologs.

In one aspect the detecting all contigs encoding the Cas1 protein isperformed by GenemarkS which a gene prediction program as furtherdescribed in “GeneMarkS: a self-training method for prediction of genestarts in microbial genomes. Implications for finding sequence motifs inregulatory regions.” John Besemer, Alexandre Lomsadze and MarkBorodovsky, Nucleic Acids Research (2001) 29, pp 2607-2618, hereinincorporated by reference.

In one aspect the identifying all predicted protein coding genes iscarried out by comparing the identified genes with Cas protein-specificprofiles and annotating them according to NCBI Conserved Domain Database(CDD) which is a protein annotation resource that consists of acollection of well-annotated multiple sequence alignment models forancient domains and full-length proteins. These are available asposition-specific score matrices (PSSMs) for fast identification ofconserved domains in protein sequences via RPS-BLAST. CDD contentincludes NCBI-curated domains, which use 3D-structure information toexplicitly define domain boundaries and provide insights intosequence/structure/function relationships, as well as domain modelsimported from a number of external source databases (Pfam, SMART, COG,PRK, TIGRFAM). In a further aspect, CRISPR arrays were predicted using aPILER-CR program which is a public domain software for finding CRISPRrepeats as described in “PILER-CR: fast and accurate identification ofCRISPR repeats”, Edgar, R. C., BMC Bioinformatics, January 20;8:18(2007), herein incorporated by reference.

In a further aspect, the case by case analysis is performed usingPSI-BLAST (Position-Specific Iterative Basic Local Alignment SearchTool). PSI-BLAST derives a position-specific scoring matrix (PSSM) orprofile from the multiple sequence alignment of sequences detected abovea given score threshold using protein-protein BLAST. This PSSM is usedto further search the database for new matches, and is updated forsubsequent iterations with these newly detected sequences. Thus,PSI-BLAST provides a means of detecting distant relationships betweenproteins.

In another aspect, the case by case analysis is performed using HHpred,a method for sequence database searching and structure prediction thatis as easy to use as BLAST or PSI-BLAST and that is at the same timemuch more sensitive in finding remote homologs. In fact, HHpred'ssensitivity is competitive with the most powerful servers for structureprediction currently available. HHpred is the first server that is basedon the pairwise comparison of profile hidden Markov models (HMMs).Whereas most conventional sequence search methods search sequencedatabases such as UniProt or the NR, HHpred searches alignmentdatabases, like Pfam or SMART. This greatly simplifies the list of hitsto a number of sequence families instead of a clutter of singlesequences. All major publicly available profile and alignment databasesare available through HHpred. HHpred accepts a single query sequence ora multiple alignment as input. Within only a few minutes it returns thesearch results in an easy-to-read format similar to that of PSI-BLAST.Search options include local or global alignment and scoring secondarystructure similarity. HHpred can produce pairwise query-templatesequence alignments, merged query-template multiple alignments (e.g. fortransitive searches), as well as 3D structural models calculated by theMODELLER software from HHpred alignments.

The term “nucleic acid-targeting system”, wherein nucleic acid is DNA orRNA, and in some aspects may also refer to DNA-RNA hybrids orderivatives thereof, refers collectively to transcripts and otherelements involved in the expression of or directing the activity of DNAor RNA-targeting CRISPR-associated (“Cas”) genes, which may includesequences encoding a DNA or RNA-targeting Cas protein and a DNA orRNA-targeting guide RNA comprising a CRISPR RNA (crRNA) sequence and (insome but not all systems) a trans-activating CRISPR/Cas system RNA(tracrRNA) sequence, or other sequences and transcripts from a DNA orRNA-targeting CRISPR locus. In general, a RNA-targeting system ischaracterized by elements that promote the formation of a DNA orRNA-targeting complex at the site of a target DNA or RNA sequence. Inthe context of formation of a DNA or RNA-targeting complex, “targetsequence” refers to a DNA or RNA sequence to which a DNA orRNA-targeting guide RNA is designed to have complementarity, wherehybridization between a target sequence and a RNA-targeting guide RNApromotes the formation of a RNA-targeting complex. In some embodiments,a target sequence is located in the nucleus or cytoplasm of a cell.

In an aspect of the invention, novel DNA targeting systems also referredto as DNA-targeting CRISPR/Cas or the CRISPR-Cas DNA-targeting system ofthe present application are based on identified Type V (e.g. subtypeV-A, subtype V-B and subtype V-C) Cas proteins which do not require thegeneration of customized proteins to target specific DNA sequences butrather a single effector protein or enzyme can be programmed by a RNAmolecule to recognize a specific DNA target, in other words the enzymecan be recruited to a specific DNA target using said RNA molecule.Aspects of the invention particularly relate to DNA targeting RNA-guidedC2c1 or C2c3 CRISPR systems.

In an aspect of the invention, novel RNA targeting systems also referredto as RNA- or RNA-targeting CRISPR/Cas or the CRISPR-Cas systemRNA-targeting system of the present application are based on identifiedType VI Cas proteins which do not require the generation of customizedproteins to target specific RNA sequences but rather a single enzyme canbe programmed by a RNA molecule to recognize a specific RNA target, inother words the enzyme can be recruited to a specific RNA target usingsaid RNA molecule.

The nucleic acids-targeting systems, the vector systems, the vectors andthe compositions described herein may be used in various nucleicacids-targeting applications, altering or modifying synthesis of a geneproduct, such as a protein, nucleic acids cleavage, nucleic acidsediting, nucleic acids splicing; trafficking of target nucleic acids,tracing of target nucleic acids, isolation of target nucleic acids,visualization of target nucleic acids, etc.

As used herein, a Cas protein or a CRISPR enzyme refers to any of theproteins presented in the new classification of CRISPR-Cas systems. Inan advantageous embodiment, the present invention encompasses effectorproteins identified in a Type V CRISPR-Cas loci, noting that aCpf1-encodes a loci denoted as subtype V-A. Presently, the subtype V-Aloci encompasses cas1, cas2, a distinct gene denoted cpf1 and a CRISPRarray. Cpf1 (CRISPR-associated protein Cpf1, subtype PREFRAN) is a largeprotein (about 1300 amino acids) that contains a RuvC-like nucleasedomain homologous to the corresponding domain of Cas9 along with acounterpart to the characteristic arginine-rich cluster of Cas9.However, Cpf1 lacks the HNH nuclease domain that is present in all Cas9proteins, and the RuvC-like domain is contiguous in the Cpf1 sequence,in contrast to Cas9 where it contains long inserts including the HNHdomain. C2c1 and C2c3 are related to Cpf1 in that they are also encodedby Type II Class V CRIPSR loci. Accordingly, in certain embodiments, theCRISPR-Cas enzyme comprises only a RuvC-like nuclease domain. The Cpf1gene is found in several diverse bacterial genomes, typically in thesame locus with cas1, cas2, and cas4 genes and a CRISPR cassette (forexample, FNFX1_1431-FNFX1_1428 of Francisella cf. novicida Fx1). Thus,the layout of Cpf1 appears to be similar to that of type II-B.Furthermore, similar to Cas9, the Cpf1 protein contains a readilyidentifiable C-terminal region that is homologous to the transposonORF-B and includes an active RuvC-like nuclease, an arginine-richregion, and a Zn finger (absent in Cas9). However, unlike Cas9, Cpf1 isalso present in several genomes without a CRISPR-Cas context and itsrelatively high similarity with ORF-B suggests that it might be atransposon component. It was suggested that if this was a genuineCRISPR-Cas system and Cpf1 is a functional analog of Cas9 it would be anovel CRISPR-Cas type, namely type V (See Annotation and Classificationof CRISPR-Cas Systems. Makarova K S, Koonin E V. Methods Mol Biol. 2015;1311:47-75). However, as described herein, Cpf1 is denoted to be insubtype V-A to distinguish it from C2c1p and C2c3p which do not have anidentical domain structure and are hence denoted to be in subtype V-Band V-C, respectively.

In an advantageous embodiment, the present invention encompassescompositions and systems comprising effector proteins identified in aC2c1 loci denoted as subtype V-B. Herein, C2c1 refers to Class 2candidate 1. All C2c1 loci encode a Cas1-Cas4 fusion, Cas2, and thelarge protein Applicants denote as C2c1p, and typically, are adjacent toa CRISPR array.

In an advantageous embodiment, the present invention encompassescompositions and systems comprising effector proteins identified in aC2c3 loci denoted as subtype V-C. Herein, C2c3 refers to Class 2candidate 3. C2c3 loci encode Cas1 and the large protein denoted C2c3p.

In an advantageous embodiment, the present invention encompasseseffector proteins identified in a Type VI CRISPR-Cas loci, e.g. the C2c2loci. Herein, C2c2 refers to Class 2 candidate 2. The C2c2 lociencompass cas1 and cas2 genes along with the large protein Applicantsdenote as C2c2p, and a CRISPR array; however, unlike C2c1p, C2c2p isoften encoded next to a CRISPR array but not cas1-cas2 (compare FIG. 9and FIG. 15).

Aspects of the invention also encompass methods and uses of thecompositions and systems described herein in genome engineering, e.g.for altering or manipulating the expression of one or more genes or theone or more gene products, in prokaryotic or eukaryotic cells, in vitro,in vivo or ex vivo.

In embodiments of the invention the terms guide sequence and guide RNAare used interchangeably as in foregoing cited documents such as WO2014/093622 (PCT/US2013/074667). In general, a guide sequence is anypolynucleotide sequence having sufficient complementarity with a targetpolynucleotide sequence to hybridize with the target sequence and directsequence-specific binding of a CRISPR complex to the target sequence. Insome embodiments, the degree of complementarity between a guide sequenceand its corresponding target sequence, when optimally aligned using asuitable alignment algorithm, is about or more than about 50%, 60%, 75%,80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may bedetermined with the use of any suitable algorithm for aligningsequences, non-limiting example of which include the Smith-Watermanalgorithm, the Needleman-Wunsch algorithm, algorithms based on theBurrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW,Clustal X, BLAT, Novoalign (Novocraft Technologies; available atwww.novocraft.com), ELAND (Illumina, San Diego, Calif.), SOAP (availableat soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). Insome embodiments, a guide sequence is about or more than about 5, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In someembodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30,25, 20, 15, 12, or fewer nucleotides in length. Preferably the guidesequence is 10-30 nucleotides long. The ability of a guide sequence todirect sequence-specific binding of a CRISPR complex to a targetsequence may be assessed by any suitable assay. For example, thecomponents of a CRISPR system sufficient to form a CRISPR complex,including the guide sequence to be tested, may be provided to a hostcell having the corresponding target sequence, such as by transfectionwith vectors encoding the components of the CRISPR sequence, followed byan assessment of preferential cleavage within the target sequence, suchas by Surveyor assay as described herein. Similarly, cleavage of atarget polynucleotide sequence may be evaluated in a test tube byproviding the target sequence, components of a CRISPR complex, includingthe guide sequence to be tested and a control guide sequence differentfrom the test guide sequence, and comparing binding or rate of cleavageat the target sequence between the test and control guide sequencereactions. Other assays are possible, and will occur to those skilled inthe art. A guide sequence may be selected to target any target sequence.In some embodiments, the target sequence is a sequence within a genomeof a cell. Exemplary target sequences include those that are unique inthe target genome.

In general, and throughout this specification, the term “vector” refersto a nucleic acid molecule capable of transporting another nucleic acidto which it has been linked. Vectors include, but are not limited to,nucleic acid molecules that are single-stranded, double-stranded, orpartially double-stranded; nucleic acid molecules that comprise one ormore free ends, no free ends (e.g., circular); nucleic acid moleculesthat comprise DNA, RNA, or both; and other varieties of polynucleotidesknown in the art. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments canbe inserted, such as by standard molecular cloning techniques. Anothertype of vector is a viral vector, wherein virally-derived DNA or RNAsequences are present in the vector for packaging into a virus (e.g.,retroviruses, replication defective retroviruses, adenoviruses,replication defective adenoviruses, and adeno-associated viruses). Viralvectors also include polynucleotides carried by a virus for transfectioninto a host cell. Certain vectors are capable of autonomous replicationin a host cell into which they are introduced (e.g., bacterial vectorshaving a bacterial origin of replication and episomal mammalianvectors). Other vectors (e.g., non-episomal mammalian vectors) areintegrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively-linked. Such vectors are referred toherein as “expression vectors.” Vectors for and that result inexpression in a eukaryotic cell can be referred to herein as “eukaryoticexpression vectors.” Common expression vectors of utility in recombinantDNA techniques are often in the form of plasmids.

Recombinant expression vectors can comprise a nucleic acid of theinvention in a form suitable for expression of the nucleic acid in ahost cell, which means that the recombinant expression vectors includeone or more regulatory elements, which may be selected on the basis ofthe host cells to be used for expression, that is operatively-linked tothe nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory element(s)in a manner that allows for expression of the nucleotide sequence (e.g.,in an in vitro transcription/translation system or in a host cell whenthe vector is introduced into the host cell).

The term “regulatory element” is intended to include promoters,enhancers, internal ribosomal entry sites (IRES), and other expressioncontrol elements (e.g., transcription termination signals, such aspolyadenylation signals and poly-U sequences). Such regulatory elementsare described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).Regulatory elements include those that direct constitutive expression ofa nucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). A tissue-specific promoter maydirect expression primarily in a desired tissue of interest, such asmuscle, neuron, bone, skin, blood, specific organs (e.g., liver,pancreas), or particular cell types (e.g., lymphocytes). Regulatoryelements may also direct expression in a temporal-dependent manner, suchas in a cell-cycle dependent or developmental stage-dependent manner,which may or may not also be tissue or cell-type specific. In someembodiments, a vector comprises one or more pol III promoter (e.g., 1,2, 3, 4, 5, or more pol III promoters), one or more pol II promoters(e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol Ipromoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), orcombinations thereof. Examples of pol III promoters include, but are notlimited to, U6 and H1 promoters. Examples of pol II promoters include,but are not limited to, the retroviral Rous sarcoma virus (RSV) LTRpromoter (optionally with the RSV enhancer), the cytomegalovirus (CMV)promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al,Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductasepromoter, the β-actin promoter, the phosphoglycerol kinase (PGK)promoter, and the EF1α promoter. Also encompassed by the term“regulatory element” are enhancer elements, such as WPRE; CMV enhancers;the R-U5′ segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p.466-472, 1988); SV40 enhancer; and the intron sequence between exons 2and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p.1527-31, 1981). It will be appreciated by those skilled in the art thatthe design of the expression vector can depend on such factors as thechoice of the host cell to be transformed, the level of expressiondesired, etc. A vector can be introduced into host cells to therebyproduce transcripts, proteins, or peptides, including fusion proteins orpeptides, encoded by nucleic acids as described herein (e.g., clusteredregularly interspersed short palindromic repeats (CRISPR) transcripts,proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.).

Advantageous vectors include lentiviruses and adeno-associated viruses,and types of such vectors can also be selected for targeting particulartypes of cells.

As used herein, the term “crRNA” or “guide RNA” or “single guide RNA” or“sgRNA” or “one or more nucleic acid components” of a Type V or Type VICRISPR-Cas locus effector protein comprises any polynucleotide sequencehaving sufficient complementarity with a target nucleic acid sequence tohybridize with the target nucleic acid sequence and directsequence-specific binding of a nucleic acid-targeting complex to thetarget nucleic acid sequence. In some embodiments, the degree ofcomplementarity, when optimally aligned using a suitable alignmentalgorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%,95%, 97.5%, 99%, or more. Optimal alignment may be determined with theuse of any suitable algorithm for aligning sequences, non-limitingexample of which include the Smith-Waterman algorithm, theNeedleman-Wunsch algorithm, algorithms based on the Burrows-WheelerTransform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X,BLAT, Novoalign (Novocraft Technologies; available atwww.novocraft.com), ELAND (Illumina, San Diego, Calif.), SOAP (availableat soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).The ability of a guide sequence (within a nucleic acid-targeting guideRNA) to direct sequence-specific binding of a nucleic acid −targetingcomplex to a target nucleic acid sequence may be assessed by anysuitable assay. For example, the components of a nucleic acid-targetingCRISPR system sufficient to form a nucleic acid-targeting complex,including the guide sequence to be tested, may be provided to a hostcell having the corresponding target nucleic acid sequence, such as bytransfection with vectors encoding the components of the nucleicacid-targeting complex, followed by an assessment of preferentialtargeting (e.g., cleavage) within the target nucleic acid sequence, suchas by Surveyor assay as described herein. Similarly, cleavage of atarget nucleic acid sequence may be evaluated in a test tube byproviding the target nucleic acid sequence, components of a nucleicacid-targeting complex, including the guide sequence to be tested and acontrol guide sequence different from the test guide sequence, andcomparing binding or rate of cleavage at the target sequence between thetest and control guide sequence reactions. Other assays are possible,and will occur to those skilled in the art. A guide sequence, and hencea nucleic acid-targeting guide RNA may be selected to target any targetnucleic acid sequence. The target sequence may be DNA. The targetsequence may be any RNA sequence. In some embodiments, the targetsequence may be a sequence within a RNA molecule selected from the groupconsisting of messenger RNA (mRNA), pre-mRNA, ribosomal RNA (rRNA),transfer RNA (tRNA), micro-RNA (miRNA), small interfering RNA (siRNA),small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), double strandedRNA (dsRNA), non coding RNA (ncRNA), long non-coding RNA (lncRNA), andsmall cytoplasmatic RNA (scRNA). In some preferred embodiments, thetarget sequence may be a sequence within a RNA molecule selected fromthe group consisting of mRNA, pre-mRNA, and rRNA. In some preferredembodiments, the target sequence may be a sequence within a RNA moleculeselected from the group consisting of ncRNA, and lncRNA. In some morepreferred embodiments, the target sequence may be a sequence within anmRNA molecule or a pre-mRNA molecule.

In some embodiments, a nucleic acid-targeting guide RNA is selected toreduce the degree secondary structure within the RNA-targeting guideRNA. In some embodiments, about or less than about 75%, 50%, 40%, 30%,25%, 20%, 15%, 10%, 5%, 1%, or fewer of the nucleotides of the nucleicacid-targeting guide RNA participate in self-complementary base pairingwhen optimally folded. Optimal folding may be determined by any suitablepolynucleotide folding algorithm. Some programs are based on calculatingthe minimal Gibbs free energy. An example of one such algorithm ismFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981),133-148). Another example folding algorithm is the online webserverRNAfold, developed at Institute for Theoretical Chemistry at theUniversity of Vienna, using the centroid structure prediction algorithm(see e.g., A. R Gruber et al., 2008, Cell 106(1): 23-24; and P A Carrand G M Church, 2009, Nature Biotechnology 27(12): 1151-62).

In certain embodiments, a guide RNA or crRNA may comprise, consistessentially of, or consist of a direct repeat (DR) sequence and a guidesequence or spacer sequence. In certain embodiments, the guide RNA orcrRNA may comprise, consist essentially of, or consist of a directrepeat sequence fused or linked to a guide sequence or spacer sequence.In certain embodiments, the direct repeat sequence may be locatedupstream (i.e., 5′) from the guide sequence or spacer sequence. In otherembodiments, the direct repeat sequence may be located downstream (i.e.,3′) from the guide sequence or spacer sequence.

In certain embodiments, the crRNA comprises a stem loop, preferably asingle stem loop. In certain embodiments, the direct repeat sequenceforms a stem loop, preferably a single stem loop.

In certain embodiments, the spacer length of the guide RNA is from 15 to35 nt. In certain embodiments, the spacer length of the guide RNA is atleast 15 nucleotides. In certain embodiments, the spacer length is from15 to 17 nt, e.g., 15, 16, or 17 nt, from 17 to 20 nt, e.g., 17, 18, 19,or 20 nt, from 20 to 24 nt, e.g., 20, 21, 22, 23, or 24 nt, from 23 to25 nt, e.g., 23, 24, or 25 nt, from 24 to 27 nt, e.g., 24, 25, 26, or 27nt, from 27-30 nt, e.g., 27, 28, 29, or 30 nt, from 30-35 nt, e.g., 30,31, 32, 33, 34, or 35 nt, or 35 nt or longer.

The “tracrRNA” sequence or analogous terms includes any polynucleotidesequence that has sufficient complementarity with a crRNA sequence tohybridize. In general, degree of complementarity is with reference tothe optimal alignment of the sca sequence and tracr sequence, along thelength of the shorter of the two sequences. Optimal alignment may bedetermined by any suitable alignment algorithm, and may further accountfor secondary structures, such as self-complementarity within either thesca sequence or tracr sequence. In some embodiments, the degree ofcomplementarity between the tracr sequence and sca sequence along thelength of the shorter of the two when optimally aligned is about or morethan about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, orhigher. In certain embodiments, the tracrRNA may not be required.

Applicants also perform a challenge experiment to verify the DNAtargeting and cleaving capability of a Type V protein such as C2c1 orC2c3. This experiment closely parallels similar work in E. coli for theheterologous expression of StCas9 (Sapranauskas, R. et al. Nucleic AcidsRes 39, 9275-9282 (2011)). Applicants introduce a plasmid containingboth a PAM and a resistance gene into the heterologous E. coli, and thenplate on the corresponding antibiotic. If there is DNA cleavage of theplasmid, Applicants observe no viable colonies.

In further detail, the assay is as follows for a DNA target. Two E. colistrains are used in this assay. One carries a plasmid that encodes theendogenous effector protein locus from the bacterial strain. The otherstrain carries an empty plasmid (e.g. pACYC184, control strain). Allpossible 7 or 8 bp PAM sequences are presented on an antibioticresistance plasmid (pUC19 with ampicillin resistance gene). The PAM islocated next to the sequence of proto-spacer 1 (the DNA target to thefirst spacer in the endogenous effector protein locus). Two PAMlibraries were cloned. One has a 8 random bp 5′ of the proto-spacer(e.g. total of 65536 different PAM sequences=complexity). The otherlibrary has 7 random bp 3′ of the proto-spacer (e.g. total complexity is16384 different PAMs). Both libraries were cloned to have in average 500plasmids per possible PAM. Test strain and control strain weretransformed with 5′ PAM and 3′ PAM library in separate transformationsand transformed cells were plated separately on ampicillin plates.Recognition and subsequent cutting/rnterference with the plasmid rendersa cell vulnerable to ampicillin and prevents growth. Approximately 12hafter transformation, all colonies formed by the test and controlstrains where harvested and plasmid DNA was isolated. Plasmid DNA wasused as template for PCR amplification and subsequent deep sequencing.Representation of all PAMs in the untransformed libraries showed theexpected representation of PAMs in transformed cells. Representation ofall PAMs found in control strains showed the actual representation.Representation of all PAMs in test strain showed which PAMs are notrecognized by the enzyme and comparison to the control strain allowsextracting the sequence of the depleted PAM.

In some embodiments of CRISPR-Cas9 systems, the degree ofcomplementarity between the tracrRNA sequence and crRNA sequence isalong the length of the shorter of the two when optimally aligned. Asdescribed herein, in embodiments of the present invention, the tracrRNAis not required. In some embodiments of previously described CRISPR-Cassystems (e.g. CRISPR-Cas9 systems), chimeric synthetic guide RNAs(sgRNAs) designs may incorporate at least 12 bp of duplex structurebetween the crRNA and tracrRNA, however in the Cpf1 CRISPR systemsdescribed herein such chimeric RNAs (chi-RNAs) are not possible as thesystem does not utilize a tracrRNA.

In certain embodiments of the invention, the mature crRNAs include asequence element derived from the CRISPR locus repeat a (the 5′ tag)sequence that is important for function. See, Marraffini et al., 28 Jan.2010, Nature Letters 463(568-572), which is incorporated by reference.

In some embodiments, the degree of complementarity between the tracrRNAsequence and crRNA sequence along the length of the shorter of the twowhen optimally aligned is about or more than about 25%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher. In some embodiments, thetracr sequence is about or more than about 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides inlength. In some embodiments, the tracr sequence and crRNA sequence arecontained within a single transcript, such that hybridization betweenthe two produces a transcript having a secondary structure, such as ahairpin. In an embodiment of the invention, the transcript ortranscribed polynucleotide sequence has at least two or more hairpins.In preferred embodiments, the transcript has two, three, four or fivehairpins. In a further embodiment of the invention, the transcript hasat most five hairpins. In a hairpin structure the portion of thesequence 5′ of the final “N” and upstream of the loop corresponds to thetracr mate sequence, and the portion of the sequence 3′ of the loopcorresponds to the tracr sequence. In certain embodiments, the tracrRNAmay not be required.

In some embodiments of previously described CRISPR-Cas systems (e.g.CRISPR-Cas9 systems), chimeric synthetic guide RNAs (sgRNAs) designs mayincorporate at least 12 bp of duplex structure between the crRNA andtracrRNA, however in the Cpf1 CRISPR systems such chimeric RNAs(chi-RNAs) are not possible as the system does not utilize a tracrRNA.

For minimization of toxicity and off-target effect, it will be importantto control the concentration of nucleic acid-targeting guide RNAdelivered. Optimal concentrations of nucleic acid-targeting guide RNAcan be determined by testing different concentrations in a cellular ornon-human eukaryote animal model and using deep sequencing to analyzethe extent of modification at potential off-target genomic loci. Theconcentration that gives the highest level of on-target modificationwhile minimizing the level of off-target modification should be chosenfor in vivo delivery. The nucleic acid-targeting system is derivedadvantageously from a Type V/Type VI CRISPR system. In some embodiments,one or more elements of a nucleic acid-targeting system is derived froma particular organism comprising an endogenous RNA-targeting system. Inpreferred embodiments of the invention, the RNA-targeting system is aType V CRISPR system. In particular embodiments, the Type VRNA-targeting Cas enzyme is C2c1 or C2c3. Non-limiting examples of Casproteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8,Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1,Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3,Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX,Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologues thereof, ormodified versions thereof. In embodiments, the Type V protein such asC2c1 or C2c3 as referred to herein also encompasses a homologue or anorthologue of a Type V protein such as C2c1 or C2c3. The terms“orthologue” (also referred to as “ortholog” herein) and “homologue”(also referred to as “homolog” herein) are well known in the art. Bymeans of further guidance, a “homologue” of a protein as used herein isa protein of the same species which performs the same or a similarfunction as the protein it is a homologue of. Homologous proteins maybut need not be structurally related, or are only partially structurallyrelated. An “orthologue” of a protein as used herein is a protein of adifferent species which performs the same or a similar function as theprotein it is an orthologue of. Orthologous proteins may but need not bestructurally related, or are only partially structurally related. Inparticular embodiments, the homologue or orthologue of a Type V proteinsuch as C2c1 or C2c3 as referred to herein has a sequence homology oridentity of at least 80%, more preferably at least 85%, even morepreferably at least 90%, such as for instance at least 95% with a Type Vprotein such as C2c1 or C2c3. In further embodiments, the homologue ororthologue of a Type V protein such as C2c1 or C2c3 as referred toherein has a sequence identity of at least 80%, more preferably at least85%, even more preferably at least 90%, such as for instance at least95% with the wild type Type V protein such as C2c1 or C2c3.

In an embodiment, the Type V RNA-targeting Cas protein may be a C2c1 orC2c3 ortholog of an organism of a genus which includes but is notlimited to Corynebacter, Sutterella Legionella Treponema Filifactor,Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides,Flavivirus, Flavobacterium, Sphaerochaeta Azospirillum,Gluconacetobacter, Neisseria Roseburia Parvibaculum, Staphylococcus,Nitratifractor, Mycoplasma and Campylobacter. Species of organism ofsuch a genus can be as otherwise herein discussed.

Some methods of identifying orthologs of CRISPR-Cas system enzymes mayinvolve identifying tracr sequences in genomes of interest.Identification of tracr sequences may relate to the following steps:Search for the direct repeats or tracr mate sequences in a database toidentify a CRISPR region comprising a CRISPR enzyme. Search forhomologous sequences in the CRISPR region flanking the CRISPR enzyme inboth the sense and antisense directions. Look for transcriptionalterminators and secondary structures. Identify any sequence that is nota direct repeat or a tracr mate sequence but has more than 50% identityto the direct repeat or tracr mate sequence as a potential tracrsequence. Take the potential tracr sequence and analyze fortranscriptional terminator sequences associated therewith.

It will be appreciated that any of the functionalities described hereinmay be engineered into CRISPR enzymes from other orthologs, includingchimeric enzymes comprising fragments from multiple orthologs. Examplesof such orthologs are described elsewhere herein. Thus, chimeric enzymesmay comprise fragments of CRISPR enzyme orthologs of an organism whichincludes but is not limited to Corynebacter, Sutterelk LegionelkaTreponema, Fiifactor, Eubacterium, Streptococcus, Lactobacillus,Mycoplasma Bacteroides, Flavivirus, Flavobacterium, Sphaerochaeta,Azospirillum, Gluconacetobacter, Neisseria Roseburia Parvibaculum,Staphylococcus, Nitratifractor, Mycoplasma and Campylobacter. A chimericenzyme can comprise a first fragment and a second fragment, and thefragments can be of CRISPR enzyme orthologs of organisms of genusesherein mentioned or of species herein mentioned; advantageously thefragments are from CRISPR enzyme orthologs of different species.

In embodiments, the Type V/Type VI RNA-targeting effector protein, inparticular the C2c1/C2c3/ protein as referred to herein also encompassesa functional variant of C2c1/C2c3 or a homologue or an orthologuethereof. A “functional variant” of a protein as used herein refers to avariant of such protein which retains at least partially the activity ofthat protein. Functional variants may include mutants (which may beinsertion, deletion, or replacement mutants), including polymorphs, etc.Also included within functional variants are fusion products of suchprotein with another, usually unrelated, nucleic acid, protein,polypeptide or peptide. Functional variants may be naturally occurringor may be man-made. Advantageous embodiments can involve engineered ornon-naturally occurring Type V/Type VI RNA-targeting effector protein,e.g., C2c1/C2c3 or an ortholog or homolog thereof.

In an embodiment, nucleic acid molecule(s) encoding the Type V/Type VIRNA-targeting effector protein, in particular C2c1/C2c3 or an orthologor homolog thereof, may be codon-optimized for expression in aneukaryotic cell. A eukaryote can be as herein discussed. Nucleic acidmolecule(s) can be engineered or non-naturally occurring.

In an embodiment, the Type V/Type VI RNA-targeting effector protein, inparticular C2c1/C2c3 or an ortholog or homolog thereof, may comprise oneor more mutations (and hence nucleic acid molecule(s) coding for samemay have mutation(s). The mutations may be artificially introducedmutations and may include but are not limited to one or more mutationsin a catalytic domain. Examples of catalytic domains with reference to aCas9 enzyme may include but are not limited to RuvC I, RuvC II, RuvC IIIand HNH domains.

In an embodiment, the Type V/Type VI protein such as C2c1 or C2c3 or anortholog or homolog thereof, may comprise one or more mutations. Themutations may be artificially introduced mutations and may include butare not limited to one or more mutations in a catalytic domain. Examplesof catalytic domains with reference to a Cas enzyme may include but arenot limited to RuvC I, RuvC II, RuvC III, HNH domains, and HEPN domains.

In an embodiment, the Type V/Type VI protein such as C2c1 or C2c3 or anortholog or homolog thereof, may be used as a generic nucleic acidbinding protein with fusion to or being operably linked to a functionaldomain. Exemplary functional domains may include but are not limited totranslational initiator, translational activator, translationalrepressor, nucleases, in particular ribonucleases, a spliceosome, beads,a light inducible/controllable domain or a chemicallyinducible/controllable domain.

In some embodiments, the unmodified nucleic acid-targeting effectorprotein may have cleavage activity. In some embodiments, theRNA-targeting effector protein may direct cleavage of one or bothnucleic acid (DNA or RNA) strands at the location of or near a targetsequence, such as within the target sequence and/or within thecomplement of the target sequence or at sequences associated with thetarget sequence. In some embodiments, the nucleic acid-targeting Casprotein may direct cleavage of one or both DNA or RNA strands withinabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, ormore base pairs from the first or last nucleotide of a target sequence.In some embodiments, the cleavage may be blunt, i.e., generating bluntends. In some embodiments, the cleavage may be staggered, i.e.,generating sticky ends. In some embodiments, the cleavage may be astaggered cut with a 5′ overhang, e.g., a 5′ overhang of 1 to 5nucleotides. In some embodiments, the cleavage may be a staggered cutwith a 3′ overhang, e.g., a 3′ overhang of 1 to 5 nucleotides. In someembodiments, a vector encodes a nucleic acid-targeting Cas protein thatmay be mutated with respect to a corresponding wild-type enzyme suchthat the mutated nucleic acid-targeting Cas protein lacks the ability tocleave one or both DNA or RNA strands of a target polynucleotidecontaining a target sequence. As a further example, two or morecatalytic domains of Cas (RuvC I, RuvC IL, and RuvC III or the HNHdomain, or HEPN domain) may be mutated to produce a mutated Cassubstantially lacking all RNA cleavage activity. As described herein,corresponding catalytic domains of a C2c1 or C2c3 effector protein mayalso be mutated to produce a mutated C2c1 or C2c3 effector proteinlacking all DNA cleavage activity or having substantially reduced DNAcleavage activity. In some embodiments, a nucleic acid-targetingeffector protein may be considered to substantially lack all RNAcleavage activity when the RNA cleavage activity of the mutated enzymeis about no more than 25%, 10/a, 5%, 1%, 0.1%, 0.01%, or less of thenucleic acid cleavage activity of the non-mutated form of the enzyme; anexample can be when the nucleic acid cleavage activity of the mutatedform is nil or negligible as compared with the non-mutated form. Aneffector protein may be identified with reference to the general classof enzymes that share homology to the biggest nuclease with multiplenuclease domains from the Type V/Type VI CRISPR system. Most preferably,the effector protein is a Type V/Type VI protein such as C2c1/C2c3. Byderived, Applicants mean that the derived enzyme is largely based, inthe sense of having a high degree of sequence homology with, a wildtypeenzyme, but that it has been mutated (modified) in some way as known inthe art or as described herein.

Again, it will be appreciated that the terms Cas and CRISPR enzyme andCRISPR protein and Cas protein are generally used interchangeably and atall points of reference herein refer by analogy to novel CRISPR effectorproteins further described in this application, unless otherwiseapparent, such as by specific reference to Cas9. As mentioned above,many of the residue numberings used herein refer to the effector proteinfrom the Type V/Type VI CRISPR locus. However, it will be appreciatedthat this invention includes many more effector proteins from otherspecies of microbes. In certain embodiments, Cas may be constitutivelypresent or inducibly present or conditionally present or administered ordelivered. Cas optimization may be used to enhance function or todevelop new functions, one can generate chimeric Cas proteins. And Casmay be used as a generic nucleic acid binding protein.

Typically, in the context of an endogenous nucleic acid-targetingsystem, formation of a nucleic acid-targeting complex (comprising aguide RNA hybridized to a target sequence and complexed with one or morenucleic acid-targeting effector proteins) results in cleavage of one orboth DNA or RNA strands in or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 50, or more base pairs from) the target sequence. As usedherein the term “sequence(s) associated with a target locus of interest”refers to sequences near the vicinity of the target sequence (e.g.within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs fromthe target sequence, wherein the target sequence is comprised within atarget locus of interest).

An example of a codon optimized sequence, is in this instance a sequenceoptimized for expression in a eukaryote, e.g., humans (i.e. beingoptimized for expression in humans), or for another eukaryote, animal ormammal as herein discussed; see, e.g., SaCas9 human codon optimizedsequence in WO 2014/093622 (PCT/JS2013/074667) as an example of a codonoptimized sequence (from knowledge in the art and this disclosure, codonoptimizing coding nucleic acid molecule(s), especially as to effectorprotein (e.g., C2c1 or C2c3) is within the ambit of the skilledartisan). Whilst this is preferred, it will be appreciated that otherexamples are possible and codon optimization for a host species otherthan human, or for codon optimization for specific organs is known. Insome embodiments, an enzyme coding sequence encoding a DNA/RNA-targetingCas protein is codon optimized for expression in particular cells, suchas eukaryotic cells. The eukaryotic cells may be those of or derivedfrom a particular organism, such as a mammal, including but not limitedto human, or non-human eukaryote or animal or mammal as hereindiscussed, e.g., mouse, rat, rabbit, dog, livestock, or non-human mammalor primate. In some embodiments, processes for modifying the germ linegenetic identity of human beings and/or processes for modifying thegenetic identity of animals which are likely to cause them sufferingwithout any substantial medical benefit to man or animal, and alsoanimals resulting from such processes, may be excluded. In general,codon optimization refers to a process of modifying a nucleic acidsequence for enhanced expression in the host cells of interest byreplacing at least one codon (e.g., about or more than about 1, 2, 3, 4,5, 10, 15, 20, 25, 50, or more codons) of the native sequence withcodons that are more frequently or most frequently used in the genes ofthat host cell while maintaining the native amino acid sequence. Variousspecies exhibit particular bias for certain codons of a particular aminoacid. Codon bias (differences in codon usage between organisms) oftencorrelates with the efficiency of translation of messenger RNA (mRNA),which is in turn believed to be dependent on, among other things, theproperties of the codons being translated and the availability ofparticular transfer RNA (tRNA) molecules. The predominance of selectedtRNAs in a cell is generally a reflection of the codons used mostfrequently in peptide synthesis. Accordingly, genes can be tailored foroptimal gene expression in a given organism based on codon optimization.Codon usage tables are readily available, for example, at the “CodonUsage Database” available at www.kazusa.orjp/codon/and these tables canbe adapted in a number of ways. See Nakamura, Y., et al. “Codon usagetabulated from the international DNA sequence databases: status for theyear 2000” Nucl. Acids Res. 28:292 (2000). Computer algorithms for codonoptimizing a particular sequence for expression in a particular hostcell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.), arealso available. In some embodiments, one or more codons (e.g., 1, 2, 3,4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encodinga DNA/RNA-targeting Cas protein corresponds to the most frequently usedcodon for a particular amino acid. As to codon usage in yeast, referenceis made to the online Yeast Genome database available atwww.yeastgenome.org/community/codon_usage.shtml, or Codon selection inyeast, Bennetzen and Hall, J. Biol Chem. 1982 Mar. 25; 257(6):3026-31.As to codon usage in plants including algae, reference is made to Codonusage in higher plants, green algae, and cyanobacteria, Campbell andGowri, Plant Physiol. 1990 January; 92(1): 1-11.; as well as Codon usagein plant genes, Murray et al, Nucleic Acids Res. 1989 Jan. 25;17(2):477-98; or Selection on the codon bias of chloroplast and cyanellegenes in different plant and algal lineages, Morton B R, J. Mol. Evol.1998 April; 46(4):449-59.

In some embodiments, a vector encodes a nucleic acid-targeting effectorprotein such as the Type V RNA-targeting effector protein, in particularC2c1 or C2c3, or an ortholog or homolog thereof comprising one or morenuclear localization sequences (NLSs), such as about or more than about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, theRNA-targeting effector protein comprises about or more than about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the amino-terminus,about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs ator near the carboxy-terminus, or a combination of these (e.g., zero orat least one or more NLS at the amino-terminus and zero or at one ormore NLS at the carboxy terminus). When more than one NLS is present,each may be selected independently of the others, such that a single NLSmay be present in more than one copy and/or in combination with one ormore other NLSs present in one or more copies. In some embodiments, anNLS is considered near the N- or C-terminus when the nearest amino acidof the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, ormore amino acids along the polypeptide chain from the N- or C-terminus.Non-limiting examples of NLSs include an NLS sequence derived from: theNLS of the SV40 virus large T-antigen, having the amino acid sequencePKKKRKV (SEQ ID NO: 1); the NLS from nucleoplasmin (e.g., thenucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ IDNO: 2)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ IDNO: 3) or RQRRNELKRSP (SEQ ID NO: 4); the hRNPA1 M9 NLS having thesequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 5); thesequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 6) ofthe IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO:7) and PPKKARED (SEQ ID NO: 8) of the myoma T protein; the sequencePQPKKKPL (SEQ ID NO: 9) of human p53; the sequence SALIKKKKKMAP (SEQ IDNO: 10) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 11) andPKQKKRK (SEQ ID NO: 12) of the influenza virus NS1; the sequenceRKLKKKIKKL (SEQ ID NO: 13) of the Hepatitis virus delta antigen; thesequence REKKKFLKRR (SEQ ID NO: 14) of the mouse Mx1 protein; thesequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 15) of the humanpoly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ IDNO: 16) of the steroid hormone receptors (human) glucocorticoid. Ingeneral, the one or more NLSs are of sufficient strength to driveaccumulation of the DNA/RNA-targeting Cas protein in a detectable amountin the nucleus of a eukaryotic cell. In general, strength of nuclearlocalization activity may derive from the number of NLSs in the nucleicacid-targeting effector protein, the particular NLS(s) used, or acombination of these factors. Detection of accumulation in the nucleusmay be performed by any suitable technique. For example, a detectablemarker may be fused to the nucleic acid-targeting protein, such thatlocation within a cell may be visualized, such as in combination with ameans for detecting the location of the nucleus (e.g., a stain specificfor the nucleus such as DAPI). Cell nuclei may also be isolated fromcells, the contents of which may then be analyzed by any suitableprocess for detecting protein, such as immunohistochemistry, Westernblot, or enzyme activity assay. Accumulation in the nucleus may also bedetermined indirectly, such as by an assay for the effect of nucleicacid-targeting complex formation (e.g., assay for DNA or RNA cleavage ormutation at the target sequence, or assay for altered gene expressionactivity affected by DNA or RNA-targeting complex formation and/or DNAor RNA-targeting Cas protein activity), as compared to a control notexposed to the nucleic acid-targeting Cas protein or nucleicacid-targeting complex, or exposed to a nucleic acid-targeting Casprotein lacking the one or more NLSs. In preferred embodiments of theherein described C2c1 or C2c3 effector protein complexes and systems,the codon optimized C2c1 or C2c3 effector proteins comprise an NLSattached to the C-terminal of the protein.

In some embodiments, one or more vectors driving expression of one ormore elements of a nucleic acid-targeting system are introduced into ahost cell such that expression of the elements of the nucleicacid-targeting system direct formation of a nucleic acid-targetingcomplex at one or more target sites. For example, a nucleicacid-targeting effector enzyme and a nucleic acid-targeting guide RNAcould each be operably linked to separate regulatory elements onseparate vectors. RNA(s) of the nucleic acid-targeting system can bedelivered to a transgenic nucleic acid-targeting effector protein animalor mammal, e.g., an animal or mammal that constitutively or inducibly orconditionally expresses nucleic acid-targeting effector protein; or ananimal or mammal that is otherwise expressing nucleic acid-targetingeffector protein or has cells containing nucleic acid-targeting effectorprotein, such as by way of prior administration thereto of a vector orvectors that code for and express in vivo nucleic acid-targetingeffector protein. Alternatively, two or more of the elements expressedfrom the same or different regulatory elements, may be combined in asingle vector, with one or more additional vectors providing anycomponents of the nucleic acid-targeting system not included in thefirst vector. Nucleic acid-targeting system elements that are combinedin a single vector may be arranged in any suitable orientation, such asone element located 5′ with respect to (“upstream” of) or 3′ withrespect to (“downstream” of) a second element. The coding sequence ofone element may be located on the same or opposite strand of the codingsequence of a second element, and oriented in the same or oppositedirection. In some embodiments, a single promoter drives expression of atranscript encoding a nucleic acid-targeting effector protein and thenucleic acid-targeting guide RNA, embedded within one or more intronsequences (e.g., each in a different intron, two or more in at least oneintron, or all in a single intron). In some embodiments, the nucleicacid-targeting effector protein and the nucleic acid-targeting guide RNAmay be operably linked to and expressed from the same promoter. Deliveryvehicles, vectors, particles, nanoparticles, formulations and componentsthereof for expression of one or more elements of a nucleicacid-targeting system are as used in the foregoing documents, such as WO2014/093622 (PCT/US2013/074667). In some embodiments, a vector comprisesone or more insertion sites, such as a restriction endonucleaserecognition sequence (also referred to as a “cloning site”). In someembodiments, one or more insertion sites (e.g., about or more than about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertion sites) are locatedupstream and/or downstream of one or more sequence elements of one ormore vectors. In some embodiments, a vector comprises an insertion siteupstream of a tracr mate sequence, and optionally downstream of aregulatory element operably linked to the tracr mate sequence, such thatfollowing insertion of a guide sequence into the insertion site and uponexpression the guide sequence directs sequence-specific binding of anucleic acid-targeting complex to a target sequence in a eukaryoticcell. In some embodiments, a vector comprises two or more insertionsites, so as to allow insertion of a guide sequence at each site. Insuch an arrangement, the two or more guide sequences may comprise two ormore copies of a single guide sequence, two or more different guidesequences, or combinations of these. When multiple different guidesequences are used, a single expression construct may be used to targetnucleic acid-targeting activity to multiple different, correspondingtarget sequences within a cell. For example, a single vector maycomprise about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,or more guide sequences. In some embodiments, about or more than about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more such guide-sequence-containingvectors may be provided, and optionally delivered to a cell. In someembodiments, a vector comprises a regulatory element operably linked toan enzyme-coding sequence encoding a nucleic acid-targeting effectorprotein. nucleic acid-targeting effector protein or nucleicacid-targeting guide RNA or RNA(s) can be delivered separately; andadvantageously at least one of these is delivered via a particle ornanoparticle complex. nucleic acid-targeting effector protein mRNA canbe delivered prior to the nucleic acid-targeting guide RNA to give timefor nucleic acid-targeting effector protein to be expressed. nucleicacid-targeting effector protein mRNA might be administered 1-12 hours(preferably around 2-6 hours) prior to the administration of nucleicacid-targeting guide RNA. Alternatively, nucleic acid-targeting effectorprotein mRNA and nucleic acid-targeting guide RNA can be administeredtogether. Advantageously, a second booster dose of guide RNA can beadministered 1-12 hours (preferably around 2-6 hours) after the initialadministration of nucleic acid-targeting effector protein mRNA+guideRNA. Additional administrations of nucleic acid-targeting effectorprotein mRNA and/or guide RNA might be useful to achieve the mostefficient levels of genome modification.

In one aspect, the invention provides methods for using one or moreelements of a nucleic acid-targeting system. The nucleic acid-targetingcomplex of the invention provides an effective means for modifying atarget DNA or RNA single or double stranded, linear or super-coiled).The nucleic acid-targeting complex of the invention has a wide varietyof utility including modifying (e.g., deleting, inserting,translocating, inactivating, activating) a target DNA or RNA in amultiplicity of cell types. As such the nucleic acid-targeting complexof the invention has a broad spectrum of applications in, e.g., genetherapy, drug screening, disease diagnosis, and prognosis. An exemplarynucleic acid-targeting complex comprises a DNA or RNA-targeting effectorprotein complexed with a guide RNA hybridized to a target sequencewithin the target locus of interest.

In one embodiment, this invention provides a method of cleaving a targetRNA. The method may comprise modifying a target RNA using a nucleicacid-targeting complex that binds to the target RNA and effect cleavageof said target RNA. In an embodiment, the nucleic acid-targeting complexof the invention, when introduced into a cell, may create a break (e.g.,a single or a double strand break) in the RNA sequence. For example, themethod can be used to cleave a disease RNA in a cell. For example, anexogenous RNA template comprising a sequence to be integrated flanked byan upstream sequence and a downstream sequence may be introduced into acell. The upstream and downstream sequences share sequence similaritywith either side of the site of integration in the RNA. Where desired, adonor RNA can be mRNA. The exogenous RNA template comprises a sequenceto be integrated (e.g., a mutated RNA). The sequence for integration maybe a sequence endogenous or exogenous to the cell. Examples of asequence to be integrated include RNA encoding a protein or a non-codingRNA (e.g., a microRNA). Thus, the sequence for integration may beoperably linked to an appropriate control sequence or sequences.Alternatively, the sequence to be integrated may provide a regulatoryfunction. The upstream and downstream sequences in the exogenous RNAtemplate are selected to promote recombination between the RNA sequenceof interest and the donor RNA. The upstream sequence is a RNA sequencethat shares sequence similarity with the RNA sequence upstream of thetargeted site for integration. Similarly, the downstream sequence is aRNA sequence that shares sequence similarity with the RNA sequencedownstream of the targeted site of integration. The upstream anddownstream sequences in the exogenous RNA template can have 75%, 80%,85%, 90%, 95%, or 100% sequence identity with the targeted RNA sequence.Preferably, the upstream and downstream sequences in the exogenous RNAtemplate have about 95%, 96%, 97%, 98%, 99%, or 100% sequence identitywith the targeted RNA sequence. In some methods, the upstream anddownstream sequences in the exogenous RNA template have about 99% or100% sequence identity with the targeted RNA sequence. An upstream ordownstream sequence may comprise from about 20 bp to about 2500 bp, forexample, about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200,2300, 2400, or 2500 bp. In some methods, the exemplary upstream ordownstream sequence have about 200 bp to about 2000 bp, about 600 bp toabout 1000 bp, or more particularly about 700 bp to about 1000 bp. Insome methods, the exogenous RNA template may further comprise a marker.Such a marker may make it easy to screen for targeted integrations.Examples of suitable markers include restriction sites, fluorescentproteins, or selectable markers. The exogenous RNA template of theinvention can be constructed using recombinant techniques (see, forexample, Sambrook et al., 2001 and Ausubel et al., 1996). In a methodfor modifying a target RNA by integrating an exogenous RNA template, abreak (e.g., double or single stranded break in double or singlestranded DNA or RNA) is introduced into the DNA or RNA sequence by thenucleic acid-targeting complex, the break is repaired via homologousrecombination with an exogenous RNA template such that the template isintegrated into the RNA target. The presence of a double-stranded breakfacilitates integration of the template. In other embodiments, thisinvention provides a method of modifying expression of a RNA in aeukaryotic cell. The method comprises increasing or decreasingexpression of a target polynucleotide by using a nucleic acid-targetingcomplex that binds to the DNA or RNA (e.g., mRNA or pre-mRNA). In somemethods, a target RNA can be inactivated to effect the modification ofthe expression in a cell. For example, upon the binding of aRNA-targeting complex to a target sequence in a cell, the target RNA isinactivated such that the sequence is not translated, the coded proteinis not produced, or the sequence does not function as the wild-typesequence does. For example, a protein or microRNA coding sequence may beinactivated such that the protein or microRNA or pre-microRNA transcriptis not produced. The target RNA of a RNA-targeting complex can be anyRNA endogenous or exogenous to the eukaryotic cell. For example, thetarget RNA can be a RNA residing in the nucleus of the eukaryotic cell.The target RNA can be a sequence (e.g., mRNA or pre-mRNA) coding a geneproduct (e.g., a protein) or a non-coding sequence (e.g., ncRNA, lncRNA,tRNA, or rRNA). Examples of target RNA include a sequence associatedwith a signaling biochemical pathway, e.g., a signaling biochemicalpathway-associated RNA. Examples of target RNA include a diseaseassociated RNA. A “disease-associated” RNA refers to any RNA which isyielding translation products at an abnormal level or in an abnormalform in cells derived from a disease-affected tissues compared withtissues or cells of a non disease control. It may be a RNA transcribedfrom a gene that becomes expressed at an abnormally high level; it maybe a RNA transcribed from a gene that becomes expressed at an abnormallylow level, where the altered expression correlates with the occurrenceand/or progression of the disease. A disease-associated RNA also refersto a RNA transcribed from a gene possessing mutation(s) or geneticvariation that is directly responsible or is in linkage disequilibriumwith a gene(s) that is responsible for the etiology of a disease. Thetranslated products may be known or unknown, and may be at a normal orabnormal level. The target RNA of a RNA-targeting complex can be any RNAendogenous or exogenous to the eukaryotic cell. For example, the targetRNA can be a RNA residing in the nucleus of the eukaryotic cell. Thetarget RNA can be a sequence (e.g., mRNA or pre-mRNA) coding a geneproduct (e.g., a protein) or a non-coding sequence (e.g., ncRNA, lncRNA,tRNA, or rRNA).

In some embodiments, the method may comprise allowing a nucleicacid-targeting complex to bind to the target DNA or RNA to effectcleavage of said target DNA or RNA thereby modifying the target DNA orRNA, wherein the nucleic acid-targeting complex comprises a nucleicacid-targeting effector protein complexed with a guide RNA hybridized toa target sequence within said target DNA or RNA. In one aspect, theinvention provides a method of modifying expression of DNA or RNA in aeukaryotic cell. In some embodiments, the method comprises allowing anucleic acid-targeting complex to bind to the DNA or RNA such that saidbinding results in increased or decreased expression of said DNA or RNA;wherein the nucleic acid-targeting complex comprises a nucleicacid-targeting effector protein complexed with a guide RNA. Similarconsiderations and conditions apply as above for methods of modifying atarget DNA or RNA. In fact, these sampling, culturing andre-introduction options apply across the aspects of the presentinvention. In one aspect, the invention provides for methods ofmodifying a target DNA or RNA in a eukaryotic cell, which may be invivo, ex vivo or in vitro. In some embodiments, the method comprisessampling a cell or population of cells from a human or non-human animal,and modifying the cell or cells. Culturing may occur at any stage exvivo. The cell or cells may even be re-introduced into the non-humananimal or plant. For re-introduced cells it is particularly preferredthat the cells are stem cells.

Indeed, in any aspect of the invention, the nucleic acid-targetingcomplex may comprise a nucleic acid-targeting effector protein complexedwith a guide RNA hybridized to a target sequence.

The invention relates to the engineering and optimization of systems,methods and compositions used for the control of gene expressioninvolving DNA or RNA sequence targeting, that relate to the nucleicacid-targeting system and components thereof. In advantageousembodiments, the effector protein enzyme is a Type V protein such asC2c1 or C2c3. An advantage of the present methods is that the CRISPRsystem minimizes or avoids off-target binding and its resulting sideeffects. This is achieved using systems arranged to have a high degreeof sequence specificity for the target DNA or RNA.

In relation to a nucleic acid-targeting complex or system preferably,the tracr sequence has one or more hairpins and is 30 or morenucleotides in length, 40 or more nucleotides in length, or 50 or morenucleotides in length; the crRNA sequence is between 10 to 30nucleotides in length, the nucleic acid-targeting effector protein is aType V effector protein.

In certain embodiments, the effector protein may be an Alicyclobacillussp. C2c1p, preferably Alicyclobacillus acidoterrestris C2c1p, morepreferably Alicyclobacillus acidoterrestris ATCC 49025 C2c1p, and thecrRNA sequence may be 34 nucleotides in length, with a 5′ 14-nt directrepeat (DR) and a 20-nt spacer.

In certain embodiments, the effector protein may be a Bacillus sp.C2c1p, preferably Bacillus thermoamylovorans C2c1p, more preferablyBacillus thermoamylovorans strain B4166 C2c1p and the crRNA sequence maybe 33 nucleotides in length, with a 5′ 14-nt direct repeat (DR) and a19-nt spacer.

In certain embodiments, the effector protein may be a Type V-B locieffector protein, more particularly a C2c1p, and the crRNA sequence maybe 27 to 40 nucleotides in length, preferably 28-nt to 39-nt in length,or 29-nt to 38-nt in length, or 30-nt to 37-nt in length, morepreferably 31-nt to 36-nt in length, or 32-nt to 35-nt in length, mostpreferably 33-nt or 34-nt in length. For example, the crRNA maycomprise, consist essentially of or consist of a direct repeat (DR),preferably a 5′ DR, 12-nt to 16-nt in length, preferably 13-nt to 15-ntin length, even more preferably 14-nt in length, and a spacer 15-nt to24-nt in length, preferably 16-nt to 23-nt in length, more preferably17-nt to 22-nt in length, even more preferably 18-nt to 21-nt in length,and most preferably 19-nt or 20-nt in length.

In certain embodiments, the effector protein may be a Listeria sp.C2c2p, preferably Listeria seeligeria C2c2p, more preferably Listeriaseeligeria serovar ½b str. SLCC3954 C2c2p and the crRNA sequence may be44 to 47 nucleotides in length, with a 5′ 29-nt direct repeat (DR) and a15-nt to 18-nt spacer.

In certain embodiments, the effector protein may be a Leptotrichia sp.C2c2p, preferably Leptotrichia shahii C2c2p, more preferablyLeptotrichia shahii DSM 19757 C2c2p and the crRNA sequence may be 42 to58 nucleotides in length, with a 5′ 28-nt direct repeat (DR) and a 14-ntto 28-nt spacer.

In certain embodiments, the effector protein may be a Type VI locieffector protein, more particularly a C2c2p, and the crRNA sequence maybe 36 to 63 nucleotides in length, preferably 37-nt to 62-nt in length,or 38-nt to 61-nt in length, or 39-nt to 60-nt in length, morepreferably 40-nt to 59-nt in length, or 41-nt to 58-nt in length, mostpreferably 42-nt to 57-nt in length. For example, the crRNA maycomprise, consist essentially of or consist of a direct repeat (DR),preferably a 5′ DR, 26-nt to 31-nt in length, preferably 27-nt to 30-ntin length, even more preferably 28-nt or 29-nt in length, and a spacer10-nt to 32-nt in length, preferably 11-nt to 31-nt in length, morepreferably 12-nt to 30-nt in length, even more preferably 13-nt to 29-ntin length, and most preferably 14-nt to 28-nt in length.

In certain embodiments, the effector protein may be an Alicyclobacillussp. C2c1p, preferably Alicyclobacillus acidoterrestris C2c1p, morepreferably Alicyclobacillus acidoterrestris ATCC 49025 C2c1p, and thetracrRNA sequence may be at least 78-nt in length, e.g., may be 79-nt inlength, or may be more than 79-nt in length, e.g., may be at least 80-ntin length, or at least 90-nt in length, or at least 100-nt in length, orat least 110-nt in length, or at least 120-nt in length, or at least130-nt in length, or at least 140-nt in length, or at least 150-nt inlength, or more.

In certain embodiments, the effector protein may be a Bacillus sp.C2c1p, preferably Bacillus thermoamylovorans C2c1p, more preferablyBacillus thermoamylovorans strain B4166 C2c1p and the tracrRNA sequencemay be about 91-nt long, such as 91-nt long.

In certain embodiments, the effector protein may be a Type V-B locieffector protein, more particularly a C2c1p, and the tracrRNA sequencemay be at least 60-nt long, such as at least 65-nt in length, or atleast 70-nt in length, such as from 60-nt to 70-nt in length, or from60-nt to 70-nt in length, or from 70-nt to 80-nt in length, or from80-nt to 90-nt in length, or from 90-nt to 100-nt in length, or from100-nt to 110-nt in length, or from 110-nt to 120-nt in length, or from120-nt to 130-nt in length, or from 130-nt to 140-nt in length, or from140-nt to 150-nt in length, or more than 150-nt in length. Seeillustrative examples in FIGS. 17-21.

In certain embodiments, the effector protein may be a Type VI locieffector protein, more particularly a C2c2p, and the tracrRNA sequencemay be at least 60-nt long, such as at least 65-nt in length, or atleast 70-nt in length, such as from 60-nt to 70-nt in length, or from60-nt to 70-nt in length, or from 70-nt to 80-nt in length, or from80-nt to 90-nt in length, or from 90-nt to 100-nt in length, or from100-nt to 110-nt in length, or from 110-nt to 120-nt in length, or from120-nt to 130-nt in length, or from 130-nt to 140-nt in length, or from140-nt to 150-nt in length, or more than 150-nt in length. Seeillustrative examples in FIGS. 22-37.

In certain embodiments, the effector protein may be a Type VI locieffector protein, more particularly a C2c2p, and no tracrRNA may berequired for cleavage.

The use of two different aptamers (each associated with a distinctnucleic acid-targeting guide RNAs) allows an activator-adaptor proteinfusion and a repressor-adaptor protein fusion to be used, with differentnucleic acid-targeting guide RNAs, to activate expression of one DNA orRNA, whilst repressing another. They, along with their different guideRNAs can be administered together, or substantially together, in amultiplexed approach. A large number of such modified nucleicacid-targeting guide RNAs can be used all at the same time, for example10 or 20 or 30 and so forth, whilst only one (or at least a minimalnumber) of effector protein molecules need to be delivered, as acomparatively small number of effector protein molecules can be usedwith a large number modified guides. The adaptor protein may beassociated (preferably linked or fused to) one or more activators or oneor more repressors. For example, the adaptor protein may be associatedwith a first activator and a second activator. The first and secondactivators may be the same, but they are preferably differentactivators. Three or more or even four or more activators (orrepressors) may be used, but package size may limit the number beinghigher than 5 different functional domains. Linkers are preferably used,over a direct fusion to the adaptor protein, where two or morefunctional domains are associated with the adaptor protein. Suitablelinkers might include the GlySer linker.

It is also envisaged that the nucleic acid-targeting effectorprotein-guide RNA complex as a whole may be associated with two or morefunctional domains. For example, there may be two or more functionaldomains associated with the nucleic acid-targeting effector protein, orthere may be two or more functional domains associated with the guideRNA (via one or more adaptor proteins), or there may be one or morefunctional domains associated with the nucleic acid-targeting effectorprotein and one or more functional domains associated with the guide RNA(via one or more adaptor proteins).

The fusion between the adaptor protein and the activator or repressormay include a linker. For example, GlySer linkers GGGS (SEQ ID NO: 17)can be used. They can be used in repeats of 3 ((GGGGS)₃ (SEQ ID NO: 18))or 6 (SEQ ID NO: 19), 9 (SEQ ID NO: 20) or even 12 (SEQ ID NO: 21) ormore, to provide suitable lengths, as required. Linkers can be usedbetween the guide RNAs and the functional domain (activator orrepressor), or between the nucleic acid-targeting effector protein andthe functional domain (activator or repressor). The linkers the user toengineer appropriate amounts of “mechanical flexibility”.

The invention comprehends a nucleic acid-targeting complex comprising anucleic acid-targeting effector protein and a guide RNA, wherein thenucleic acid-targeting effector protein comprises at least one mutation,such that the nucleic acid-targeting Cas protein has no more than 5% ofthe activity of the nucleic acid-targeting Cas protein not having the atleast one mutation and, optionally, at least one or more nuclearlocalization sequences; the guide RNA comprises a guide sequence capableof hybridizing to a target sequence in a RNA of interest in a cell; andwherein: the nucleic acid-targeting effector protein is associated withtwo or more functional domains; or at least one loop of the guide RNA ismodified by the insertion of distinct RNA sequence(s) that bind to oneor more adaptor proteins, and wherein the adaptor protein is associatedwith two or more functional domains; or the nucleic acid-targetingeffector protein is associated with one or more functional domains andat least one loop of the guide RNA is modified by the insertion ofdistinct RNA sequence(s) that bind to one or more adaptor proteins, andwherein the adaptor protein is associated with one or more functionaldomains. Enzyme mutations reducing off-target effects.

In one aspect, the invention provides a non-naturally occurring orengineered CRISPR enzyme, preferably a class 2 CRISPR enzyme, preferablya Type V or VI CRISPR enzyme as described herein, such as preferably,but without limitation C2c1 or C2c3 as described herein elsewhere,having one or more mutations resulting in reduced off-target effects,i.e. improved CRISPR enzymes for use in effecting modifications totarget loci but which reduce or eliminate activity towards off-targets,such as when complexed to guide RNAs, as well as improved CRISPR enzymesfor increasing the activity of CRISPR enzymes, such as when complexedwith guide RNAs. It is to be understood that mutated enzymes asdescribed herein below may be used in any of the methods according tothe invention as described herein elsewhere. Any of the methods,products, compositions and uses as described herein elsewhere areequally applicable with the mutated CRISPR enzymes as further detailedbelow. It is to be understood, that in the aspects and embodiments asdescribed herein, when referring to or reading on C2c1 or C2c3 as theCRISPR enzyme, reconstitution of a functional CRISPR-Cas systempreferably does not require or is not dependent on a tracr sequenceand/or direct repeat is 5′ (upstream) of the guide (target or spacer)sequence.

By means of further guidance, the following particular aspects andembodiments are provided.

The inventors have surprisingly determined that modifications may bemade to CRISPR enzymes which confer reduced off-target activity comparedto unmodified CRISPR enzymes and/or increased target activity comparedto unmodified CRISPR enzymes. Thus, in certain aspects of the inventionprovided herein are improved CRISPR enzymes which may have utility in awide range of gene modifying applications. Also provided herein areCRISPR complexes, compositions and systems, as well as methods and uses,all comprising the herein disclosed modified CRISPR enzymes.

In this disclosure, the term “Cas” can mean “C2c1” or “C2c3” or a CRISPRenzyme. The terms “C2c1p” and “C2c1” are used interchangeably, and theterms “C2c3p” and “C2c3” are used interchangeably. The letter p in C2c1pand C2c3p denotes that it is a protein. In the context of this aspect ofthe invention, a C2c1 or C2c3 or CRISPR enzyme is mutated or modified,“whereby the enzyme in the CRISPR complex has reduced capability ofmodifying one or more off-target loci as compared to an unmodifiedenzyme” (or like expressions); and, when reading this specification, theterms “C2c1” or “C2c3” or “Cas” or “CRISPR enzyme” and the like aremeant to include mutated or modified C2c1 or C2c3 or Cas or CRISPRenzyme in accordance with the invention, i.e., “whereby the enzyme inthe CRISPR complex has reduced capability of modifying one or moreoff-target loci as compared to an unmodified enzyme” (or likeexpressions).

In an aspect, there is provided an engineered C2c1 or C2c3 protein asdefined herein, such as C2c1 or C2c3, wherein the protein complexes witha nucleic acid molecule comprising RNA to form a CRISPR complex, whereinwhen in the CRISPR complex, the nucleic acid molecule targets one ormore target polynucleotide loci, the protein comprises at least onemodification compared to unmodified C2c1 or unmodified C2c3 protein, andwherein the CRISPR complex comprising the modified protein has alteredactivity as compared to the complex comprising the unmodified C2c1 orunmodified C2c3 protein. It is to be understood that when referringherein to CRISPR “protein”, the C2c1 protein or the C2c3 proteinpreferably is a modified CRISPR enzyme (e.g. having increased ordecreased (or no) enzymatic activity, such as without limitationincluding C2c1 or C2c3. The term “CRISPR protein” may be usedinterchangeably with “CRISPR enzyme”, irrespective of whether the CRISPRprotein has altered, such as increased or decreased (or no) enzymaticactivity, compared to the wild type CRISPR protein.

In an aspect, the altered activity of the engineered CRISPR proteincomprises an altered binding property as to the nucleic acid moleculecomprising RNA or the target polynucleotide loci, altered bindingkinetics as to the nucleic acid molecule comprising RNA or the targetpolynucleotide loci, or altered binding specificity as to the nucleicacid molecule comprising RNA or the target polynucleotide loci comparedto off-target polynucleotide loci.

In some embodiments, the unmodified Cas has DNA cleavage activity, suchas C2c1 or C2c3. In some embodiments, the Cas directs cleavage of one orboth strands at the location of a target sequence, such as within thetarget sequence and/or within the complement of the target sequence. Insome embodiments, the Cas directs cleavage of one or both strands withinabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, ormore base pairs from the first or last nucleotide of a target sequence.In some embodiments, a vector encodes a Cas that is mutated to withrespect to a corresponding wild-type enzyme such that the mutated Caslacks the ability to cleave one or both strands of a targetpolynucleotide containing a target sequence. In some embodiments, a Casis considered to substantially lack all DNA cleavage activity when theDNA cleavage activity of the mutated enzyme is about no more than 25%,10%, 5%, 1%, 0.1%, 0.01%, or less of the DNA cleavage activity of thenon-mutated form of the enzyme; an example can be when the DNA cleavageactivity of the mutated form is nil or negligible as compared with thenon-mutated form. Thus, the Cas may comprise one or more mutations andmay be used as a generic DNA binding protein with or without fusion to afunctional domain. The mutations may be artificially introducedmutations or gain- or loss-of-function mutations. In one aspect of theinvention, the Cas enzyme may be fused to a protein, e.g., a TAG, and/oran inducible/controllable domain such as a chemicallyinducible/controllable domain. The Cas in the invention may be achimeric Cas proteins; e.g., a Cas having enhanced function by being achimera. Chimeric Cas proteins may be new Cas containing fragments frommore than one naturally occurring Cas. These may comprise fusions ofN-terminal fragment(s) of one Cas9 homolog with C-terminal fragment(s)of another Cas homolog. The Cas can be delivered into the cell in theform of mRNA. The expression of Cas can be under the control of aninducible promoter. It is explicitly an object of the invention to avoidreading on known mutations. Indeed, the phrase “whereby the enzyme inthe CRISPR complex has reduced capability of modifying one or moreoff-target loci as compared to an unmodified enzyme and/or whereby theenzyme in the CRISPR complex has increased capability of modifying theone or more target loci as compared to an unmodified enzyme” (or likeexpressions) is not intended to read upon mutations that only result ina nickase or dead Cas or known Cas9 mutations. HOWEVER, this is not tosay that the instant invention modification(s) or mutation(s) “wherebythe enzyme in the CRISPR complex has reduced capability of modifying oneor more off-target loci as compared to an unmodified enzyme and/orwhereby the enzyme in the CRISPR complex has increased capability ofmodifying the one or more target loci as compared to an unmodifiedenzyme” (or like expressions) cannot be combined with mutations thatresult in the enzyme being a nickase or dead. Such a dead enzyme can bean enhanced nucleic acid molecule binder. And such a nickase can be anenhanced nickase. For instance, changing neutral amino acid(s) in and/ornear the groove and/or other charged residues in other locations in Casthat are in close proximity to a nucleic acid (e.g., DNA, cDNA, RNA,gRNA to positive charged amino acid(s) may result in “whereby the enzymein the CRISPR complex has reduced capability of modifying one or moreoff-target loci as compared to an unmodified enzyme and/or whereby theenzyme in the CRISPR complex has increased capability of modifying theone or more target loci as compared to an unmodified enzyme”, e.g., morecutting. As this can be both enhanced on- and off-target cutting (asuper cutting C2c1 or C2c3), using such with what is known in the art asa tru-guide or tru-sgRNAs (see, e.g., Fu et al., “Improving CRISPR-Casnuclease specificity using truncated guide RNAs,” Nature Biotechnology32, 279-284 (2014) doi:10.1038/nbt.2808, Received 17 Nov. 2013, Accepted6 Jan. 2014, Published online 26 Jan. 2014, Corrected online 29 Jan.2014) to have enhanced on target activity without higher off targetcutting or for making super cutting nickases, or for combination with amutation that renders the Cas dead for a super binder.

In certain embodiments, the altered activity of the engineered C2c1 orC2c3 protein comprises increased targeting efficiency or decreasedoff-target binding. In certain embodiments, the altered activity of theengineered C2c1 or C2c3 protein comprises modified cleavage activity.

In certain embodiments, the altered activity comprises altered bindingproperty as to the nucleic acid molecule comprising RNA or the targetpolynucleotide loci, altered binding kinetics as to the nucleic acidmolecule comprising RNA or the target polynucleotide loci, or alteredbinding specificity as to the nucleic acid molecule comprising RNA orthe target polynucleotide loci compared to off-target polynucleotideloci.

In certain embodiments, the altered activity comprises increasedtargeting efficiency or decreased off-target binding. In certainembodiments, the altered activity comprises modified cleavage activity.In certain embodiments, the altered activity comprises increasedcleavage activity as to the target polynucleotide loci. In certainembodiments, the altered activity comprises decreased cleavage activityas to the target polynucleotide loci. In certain embodiments, thealtered activity comprises decreased cleavage activity as to off-targetpolynucleotide loci. In certain embodiments, the altered activitycomprises increased cleavage activity as to off-target polynucleotideloci.

Accordingly, in certain embodiments, there is increased specificity fortarget polynucleotide loci as compared to off-target polynucleotideloci. In other embodiments, there is reduced specificity for targetpolynucleotide loci as compared to off-target polynucleotide loci.

In an aspect of the invention, the altered activity of the engineeredC2c1 or C2c3 protein comprises altered helicase kinetics.

In an aspect of the invention, the engineered C2c1 or C2c3 proteincomprises a modification that alters association of the protein with thenucleic acid molecule comprising RNA, or a strand of the targetpolynucleotide loci, or a strand of off-target polynucleotide loci. Inan aspect of the invention, the engineered C2c1 or C2c3 proteincomprises a modification that alters formation of the CRISPR complex.

In certain embodiments, the modified C2c1 or C2c3 protein comprises amodification that alters targeting of the nucleic acid molecule to thepolynucleotide loci. In certain embodiments, the modification comprisesa mutation in a region of the protein that associates with the nucleicacid molecule. In certain embodiments, the modification comprises amutation in a region of the protein that associates with a strand of thetarget polynucleotide loci. In certain embodiments, the modificationcomprises a mutation in a region of the protein that associates with astrand of the off-target polynucleotide loci. In certain embodiments,the modification or mutation comprises decreased positive charge in aregion of the protein that associates with the nucleic acid moleculecomprising RNA, or a strand of the target polynucleotide loci, or astrand of off-target polynucleotide loci. In certain embodiments, themodification or mutation comprises decreased negative charge in a regionof the protein that associates with the nucleic acid molecule comprisingRNA, or a strand of the target polynucleotide loci, or a strand ofoff-target polynucleotide loci. In certain embodiments, the modificationor mutation comprises increased positive charge in a region of theprotein that associates with the nucleic acid molecule comprising RNA,or a strand of the target polynucleotide loci, or a strand of off-targetpolynucleotide loci. In certain embodiments, the modification ormutation comprises increased negative charge in a region of the proteinthat associates with the nucleic acid molecule comprising RNA, or astrand of the target polynucleotide loci, or a strand of off-targetpolynucleotide loci. In certain embodiments, the modification ormutation increases steric hindrance between the protein and the nucleicacid molecule comprising RNA, or a strand of the target polynucleotideloci, or a strand of off-target polynucleotide loci. In certainembodiments, the modification or mutation comprises a substitution ofLys, His, Arg, Glu, Asp, Ser, Gly, or Thr. In certain embodiments, themodification or mutation comprises a substitution with Gly, Ala, Ile,Glu, or Asp. In certain embodiments, the modification or mutationcomprises an amino acid substitution in a binding groove.

In as aspect, the present invention provides:

-   -   a non-naturally-occurring CRISPR enzyme as defined herein, such        as C2c1 or    -   C2c3, wherein:

the enzyme complexes with guide RNA to form a CRISPR complex,

when in the CRISPR complex, the guide RNA targets one or more targetpolynucleotide loci and the enzyme alters the polynucleotide loci, and

the enzyme comprises at least one modification,

-   -   whereby the enzyme in the CRISPR complex has reduced capability        of modifying one or more off-target loci as compared to an        unmodified enzyme, and/or whereby the enzyme in the CRISPR        complex has increased capability of modifying the one or more        target loci as compared to an unmodified enzyme.

In any such non-naturally-occurring CRISPR enzyme, the modification maycomprise modification of one or more amino acid residues of the enzyme.

In any such non-naturally-occurring CRISPR enzyme, the modification maycomprise modification of one or more amino acid residues located in aregion which comprises residues which are positively charged in theunmodified enzyme.

In any such non-naturally-occurring CRISPR enzyme, the modification maycomprise modification of one or more amino acid residues which arepositively charged in the unmodified enzyme.

In any such non-naturally-occurring CRISPR enzyme, the modification maycomprise modification of one or more amino acid residues which are notpositively charged in the unmodified enzyme.

The modification may comprise modification of one or more amino acidresidues which are uncharged in the unmodified enzyme.

The modification may comprise modification of one or more amino acidresidues which are negatively charged in the unmodified enzyme.

The modification may comprise modification of one or more amino acidresidues which are hydrophobic in the unmodified enzyme.

The modification may comprise modification of one or more amino acidresidues which are polar in the unmodified enzyme.

In certain of the above-described non-naturally-occurring CRISPRenzymes, the modification may comprise modification of one or moreresidues located in a groove.

In certain of the above-described non-naturally-occurring CRISPRenzymes, the modification may comprise modification of one or moreresidues located outside of a groove.

In certain of the above-described non-naturally-occurring CRISPRenzymes, the modification comprises a modification of one or moreresidues wherein the one or more residues comprises arginine, histidineor lysine.

In any of the above-described non-naturally-occurring CRISPR enzymes,the enzyme may be modified by mutation of said one or more residues.

In certain of the above-described non-naturally-occurring CRISPRenzymes, the enzyme is modified by mutation of said one or moreresidues, and wherein the mutation comprises substitution of a residuein the unmodified enzyme with an alanine residue.

In certain of the above-described non-naturally-occurring CRISPRenzymes, the enzyme is modified by mutation of said one or moreresidues, and wherein the mutation comprises substitution of a residuein the unmodified enzyme with aspartic acid or glutamic acid.

In certain of the above-described non-naturally-occurring CRISPRenzymes, the enzyme is modified by mutation of said one or moreresidues, and wherein the mutation comprises substitution of a residuein the unmodified enzyme with serine, threonine, asparagine orglutamine.

In certain of the above-described non-naturally-occurring CRISPRenzymes, the enzyme is modified by mutation of said one or moreresidues, and wherein the mutation comprises substitution of a residuein the unmodified enzyme with alanine, glycine, isoleucine, leucine,methionine, phenylalanine, tryptophan, tyrosine or valine.

In certain of the above-described non-naturally-occurring CRISPRenzymes, the enzyme is modified by mutation of said one or moreresidues, and wherein the mutation comprises substitution of a residuein the unmodified enzyme with a polar amino acid residue.

In certain of the above-described non-naturally-occurring CRISPRenzymes, the enzyme is modified by mutation of said one or moreresidues, and wherein the mutation comprises substitution of a residuein the unmodified enzyme with an amino acid residue which is not a polaramino acid residue.

In certain of the above-described non-naturally-occurring CRISPRenzymes, the enzyme is modified by mutation of said one or moreresidues, and wherein the mutation comprises substitution of a residuein the unmodified enzyme with a negatively charged amino acid residue.

In certain of the above-described non-naturally-occurring CRISPRenzymes, the enzyme is modified by mutation of said one or moreresidues, and wherein the mutation comprises substitution of a residuein the unmodified enzyme with an amino acid residue which is not anegatively charged amino acid residue.

In certain of the above-described non-naturally-occurring CRISPRenzymes, the enzyme is modified by mutation of said one or moreresidues, and wherein the mutation comprises substitution of a residuein the unmodified enzyme with an uncharged amino acid residue.

In certain of the above-described non-naturally-occurring CRISPRenzymes, the enzyme is modified by mutation of said one or moreresidues, and wherein the mutation comprises substitution of a residuein the unmodified enzyme with an amino acid residue which is not anuncharged amino acid residue.

In certain of the above-described non-naturally-occurring CRISPRenzymes, the enzyme is modified by mutation of said one or moreresidues, and wherein the mutation comprises substitution of a residuein the unmodified enzyme with a hydrophobic amino acid residue.

In certain of the above-described non-naturally-occurring CRISPRenzymes, the enzyme is modified by mutation of said one or moreresidues, and wherein the mutation comprises substitution of a residuein the unmodified enzyme with an amino acid residue which is not ahydrophobic amino acid residue.

In certain embodiments, the effector protein may be an Alicyclobacillussp. C2c1p, preferably Alicyclobacillus acidoterrestris C2c1p, morepreferably Alicyclobacillus acidoterrestris ATCC 49025 C2c1p. In certainembodiments, the effector protein may be a Bacillus sp. C2c1p,preferably Bacillus thermoamylovorans C2c1p, more preferably Bacillusthermoamylovorans strain B4166 C2c1p. In certain embodiments, theeffector protein may be a Type V-B loci effector protein, moreparticularly a C2c1p.

In certain embodiments, the C2c1 or C2c3 protein comprises one or morenuclear localization signal (NLS) domains. In certain embodiments, theC2c1 or C2c3 protein comprises at least two or more NLSs.

In certain embodiments, the C2c1 or C2c3 protein comprises a chimericCRISPR protein, comprising a first fragment from a first CRISPRorthologue and a second fragment from a second CIRSPR orthologue, andthe first and second CRISPR orthologues are different.

In certain embodiments, the enzyme is modified by or comprisesmodification, e.g., comprises, consists essentially of or consists ofmodification by mutation of any one of the residues listed herein or acorresponding residue in the respective orthologue; or the enzymecomprises, consists essentially of or consists of modification in anyone (single), two (double), three (triple), four (quadruple) or moreposition(s) in accordance with the disclosure throughout thisapplication, or a corresponding residue or position in the CRISPR enzymeorthologue, e.g., an enzyme comprising, consisting essentially of orconsisting of modification in any one of the C2c1 or C2c3 residuesrecited herein, or a corresponding residue or position in the CRISPRenzyme orthologue. In such an enzyme, each residue may be modified bysubstitution with an alanine residue.

Applicants recently described a method for the generation of Cas9orthologues with enhanced specificity (Slaymaker et al. 2015 “Rationallyengineered Cas9 nucleases with improved specificity”). This strategy canbe used to enhance the specificity of C2c1 or C2c3 orthologues. Primaryresidues for mutagenesis are preferably all positively charged residueswithin the RuvC domain. Additional residues are positively chargedresidues that are conserved between different orthologues.

In certain embodiments, specificity of C2c1 or C2c3 may be improved bymutating residues that stabilize the non-targeted DNA strand.

In any of the non-naturally-occurring CRISPR enzymes:

a single mismatch may exist between the target and a correspondingsequence of the one or more off-target loci; and/or

two, three or four or more mismatches may exist between the target and acorresponding sequence of the one or more off-target loci, and/or

-   -   wherein in (ii) said two, three or four or more mismatches are        contiguous.

In any of the non-naturally-occurring CRISPR enzymes the enzyme in theCRISPR complex may have reduced capability of modifying one or moreoff-target loci as compared to an unmodified enzyme and wherein theenzyme in the CRISPR complex has increased capability of modifying thesaid target loci as compared to an unmodified enzyme.

In any of the non-naturally-occurring CRISPR enzymes, when in the CRISPRcomplex the relative difference of the modifying capability of theenzyme as between target and at least one off-target locus may beincreased compared to the relative difference of an unmodified enzyme.

In any of the non-naturally-occurring CRISPR enzymes, the CRISPR enzymemay comprise one or more additional mutations, wherein the one or moreadditional mutations are in one or more catalytically active domains.

In such non-naturally-occurring CRISPR enzymes, the CRISPR enzyme mayhave reduced or abolished nuclease activity compared with an enzymelacking said one or more additional mutations.

In some such non-naturally-occurring CRISPR enzymes, the CRISPR enzymedoes not direct cleavage of one or other DNA strand at the location ofthe target sequence.

Where the CRISPR enzyme comprises one or more additional mutations inone or more catalytically active domains, the one or more additionalmutations may be in a catalytically active domain of the CRISPR enzymecomprising RuvCI, RuvCII or RuvCIII.

Without being bound by theory, in an aspect of the invention, themethods and mutations described provide for enhancing conformationalrearrangement of CRISPR enzyme domains (e.g. C2c1 or C2c3 domains) topositions that results in cleavage at on-target sits and avoidance ofthose conformational states at off-target sites. CRISPR enzymes cleavetarget DNA in a series of coordinated steps. First, the PAM-interactingdomain recognizes the PAM sequence 5′ of the target DNA. After PAMbinding, the first 10-12 nucleotides of the target sequence (seedsequence) are sampled for gRNA:DNA complementarity, a process dependenton DNA duplex separation. If the seed sequence nucleotides complementthe gRNA, the remainder of DNA is unwound and the full length of gRNAhybridizes with the target DNA strand. Nt-grooves may stabilize thenon-targeted DNA strand and facilitate unwinding through non-specificinteractions with positive charges of the DNA phosphate backbone.RNA:cDNA and CRISPR enzyme:ncDNA interactions drive DNA unwinding incompetition against cDNA:ncDNA rehybridization. Other CRISPR enzymedomains may affect the conformation of nuclease domains as well, forexample linkers connecting different domains. Accordingly, the methodsand mutations provided encompass, without limitation, RuvCI, RuvCIII,RuvCIII and linkers. Conformational changes in for instance C2c1 or C2c3brought about by target DNA binding, including seed sequenceinteraction, and interactions with the target and non-target DNA stranddetermine whether the domains are positioned to trigger nucleaseactivity. Thus, the mutations and methods provided herein demonstrateand enable modifications that go beyond PAM recognition and RNA-DNA basepairing.

In an aspect, the invention provides CRISPR nucleases as defined herein,such as C2c1 or C2c3, that comprise an improved equilibrium towardsconformations associated with cleavage activity when involved inon-target interactions and/or improved equilibrium away fromconformations associated with cleavage activity when involved inoff-target interactions. In one aspect, the invention provides Cas (e.g.C2c1 or C2c3) nucleases with improved proof-reading function, i.e. a Cas(e.g. C2c1 or C2c3) nuclease which adopts a conformation comprisingnuclease activity at an on-target site, and which conformation hasincreased unfavorability at an off-target site. Stenberg et al., Nature527(7576):110-3, doi: 10.1038/nature15544, published online 28 Oct.2015. Epub 2015 Oct. 28, used Förster resonance energy transfer FRET)experiments to detect relative orientations of the Cas9 catalyticdomains when associated with on- and off-target DNA, and which may beextrapolated to the CRISPR enzymes of the present invention (e.g. C2c1or C2c3).

The invention further provides methods and mutations for modulatingnuclease activity and/or specificity using modified guide RNAs. Asdiscussed, on-target nuclease activity can be increased or decreased.Also, off-target nuclease activity can be increased or decreased.Further, there can be increased or decreased specificity as to on-targetactivity vs. off-target activity. Modified guide RNAs include, withoutlimitation, truncated guide RNAs, dead guide RNAs, chemically modifiedguide RNAs, guide RNAs associated with functional domains, modifiedguide RNAs comprising functional domains, modified guide RNAs comprisingaptamers, modified guide RNAs comprising adapter proteins, and guideRNAs comprising added or modified loops. In some embodiments, one ormore functional domains are associated with an dead gRNA (dRNA). In someembodiments, a dRNA complex with the CRISPR enzyme directs generegulation by a functional domain at on gene locus while an gRNA directsDNA cleavage by the CRISPR enzyme at another locus. In some embodiments,dRNAs are selected to maximize selectivity of regulation for a genelocus of interest compared to off-target regulation. In someembodiments, dRNAs are selected to maximize target gene regulation andminimize target cleavage.

For the purposes of the following discussion, reference to a functionaldomain could be a functional domain associated with the CRISPR enzyme ora functional domain associated with the adaptor protein.

In the practice of the invention, loops of the gRNA may be extended,without colliding with the Cas (e.g. C2c1 or C2c3) protein by theinsertion of distinct RNA loop(s) or distinct sequence(s) that mayrecruit adaptor proteins that can bind to the distinct RNA loop(s) ordistinct sequence(s). The adaptor proteins may include but are notlimited to orthogonal RNA-binding protein/aptamer combinations thatexist within the diversity of bacteriophage coat proteins. A list ofsuch coat proteins includes, but is not limited to: Qβ, F2, GA, fr,JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI,ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s and PRR1. Theseadaptor proteins or orthogonal RNA binding proteins can further recruiteffector proteins or fusions which comprise one or more functionaldomains. In some embodiments, the functional domain may be selected fromthe group consisting of: transposase domain, integrase domain,recombinase domain, resolvase domain, invertase domain, protease domain,DNA methyltransferase domain, DNA hydroxylmethylase domain, DNAdemethylase domain, histone acetylase domain, histone deacetylasesdomain, nuclease domain, repressor domain, activator domain,nuclear-localization signal domains, transcription-regulatory protein(or transcription complex recruiting) domain, cellular uptake activityassociated domain, nucleic acid binding domain, antibody presentationdomain, histone modifying enzymes, recruiter of histone modifyingenzymes; inhibitor of histone modifying enzymes, histonemethyltransferase, histone demethylase, histone kinase, histonephosphatase, histone ribosylase, histone deribosylase, histoneubiquitinase, histone deubiquitinase, histone biotinidase and histonetail protease. In some preferred embodiments, the functional domain is atranscriptional activation domain, such as, without limitation, VP64,p65, MyoD1, HSF1, RTA, SET7/9 or a histone acetyltransferase. In someembodiments, the functional domain is a transcription repression domain,preferably KRAB. In some embodiments, the transcription repressiondomain is SID, or concatemers of SID (eg SID4X). In some embodiments,the functional domain is an epigenetic modifying domain, such that anepigenetic modifying enzyme is provided. In some embodiments, thefunctional domain is an activation domain, which may be the P65activation domain. In some embodiments, the functional domain is adeaminase, such as a cytidine deaminase. Cytidine deaminese may bedirected to a target nucleic acid to where it directs conversion ofcytidine to uridine, resulting in C to T substitutions (G to A on thecomplementary strand). In such an embodiment, nucleotide substitutionscan be effected without DNA cleavage.

In an aspect, the invention also provides methods and mutations formodulating Cas (e.g. C2c1 or C2c3) binding activity and/or bindingspecificity. In certain embodiments Cas (e.g. C2c1 or C2c3) proteinslacking nuclease activity are used. In certain embodiments, modifiedguide RNAs are employed that promote binding but not nuclease activityof a Cas (e.g. C2c1 or C2c3) nuclease. In such embodiments, on-targetbinding can be increased or decreased. Also, in such embodimentsoff-target binding can be increased or decreased. Moreover, there can beincreased or decreased specificity as to on-target binding vs.off-target binding.

In particular embodiments, a reduction of off-target cleavage is ensuredby destabilizing strand separation, more particularly by introducingmutations in the C2c1 or C2c3 enzyme decreasing the positive charge inthe DNA interacting regions (as described herein and further exemplifiedfor Cas9 by Slaymaker et al. 2016 (Science, 1; 351(6268):84-8). Infurther embodiments, a reduction of off-target cleavage is ensured byintroducing mutations into C2c1 or C2c3 enzyme which affect theinteraction between the target strand and the guide RNA sequence, moreparticularly disrupting interactions between C2c1 or C2c3 and thephosphate backbone of the target DNA strand in such a way as to retaintarget specific activity but reduce off-target activity (as describedfor Cas9 by Kleinstiver et al. 2016, Nature, 28; 529(7587):490-5). Inparticular embodiments, the off-target activity is reduced by way of amodified C2c1 (or a modified C2c3) wherein interaction with both thetarget strand and the non-target strand are modified compared towild-type C2c1 (or wild-type C2c3).

The methods and mutations which can be employed in various combinationsto increase or decrease activity and/or specificity of on-target vs.off-target activity, or increase or decrease binding and/or specificityof on-target vs. off-target binding, can be used to compensate orenhance mutations or modifications made to promote other effects. Suchmutations or modifications made to promote other effects includemutations or modification to the Cas (e.g. C2c1 or C2c3) and/or mutationor modification made to a guide RNA. In certain embodiments, the methodsand mutations are used with chemically modified guide RNAs. Examples ofguide RNA chemical modifications include, without limitation,incorporation of 2′-O-methyl (M), 2′-O-methyl 3′phosphorothioate (MS),or 2′-O-methyl 3′thioPACE (MSP) at one or more terminal nucleotides.Such chemically modified guide RNAs can comprise increased stability andincreased activity as compared to unmodified guide RNAs, thoughon-target vs. off-target specificity is not predictable. (See, Hendel,2015, Nat Biotechnol. 33(9):985-9, doi: 10.1038/nbt.3290, publishedonline 29 Jun. 2015). Chemically modified guide RNAs further include,without limitation, RNAs with phosphorothioate linkages and lockednucleic acid (LNA) nucleotides comprising a methylene bridge between the2′ and 4′ carbons of the ribose ring. The methods and mutations of theinvention are used to modulate Cas (e.g. C2c1 or C2c3) nuclease activityand/or binding with chemically modified guide RNAs.

In an aspect, the invention provides methods and mutations formodulating binding and/or binding specificity of Cas (e.g. C2c1 or C2c3)proteins according to the invention as defined herein comprisingfunctional domains such as nucleases, transcriptional activators,transcriptional repressors, and the like. For example, a Cas (e.g. C2c1or C2c3) protein can be made nuclease-null, or having altered or reducednuclease activity by introducing mutations such as for instance C2c1 orC2c3 mutations. Nuclease deficient Cas (e.g. C2c1 or C2c3) proteins areuseful for RNA-guided target sequence dependent delivery of functionaldomains. The invention provides methods and mutations for modulatingbinding of Cas (e.g. C2c1 or C2c3) proteins. In one embodiment, thefunctional domain comprises VP64, providing an RNA-guided transcriptionfactor. In another embodiment, the functional domain comprises Fok I,providing an RNA-guided nuclease activity. Mention is made of U.S. Pat.Pub. 2014/0356959, U.S. Pat. Pub. 2014/0342456, U.S. Pat. Pub.2015/0031132, and Mali, P. et al., 2013, Science 339(6121):823-6, doi:10.1126/science.1232033, published online 3 Jan. 2013, and through theteachings herein the invention comprehends methods and materials ofthese documents applied in conjunction with the teachings herein. Incertain embodiments, on-target binding is increased. In certainembodiments, off-target binding is decreased. In certain embodiments,on-target binding is decreased. In certain embodiments, off-targetbinding is increased. Accordingly, the invention also provides forincreasing or decreasing specificity of on-target binding vs. off-targetbinding of functionalized Cas (e.g. C2c1 or C2c3) binding proteins.

The use of Cas (e.g. C2c1 or C2c3) as an RNA-guided binding protein isnot limited to nuclease-null Cas (e.g. C2c1 or C2c3). Cas (e.g. C2c1 orC2c3) enzymes comprising nuclease activity can also function asRNA-guided binding proteins when used with certain guide RNAs. Forexample short guide RNAs and guide RNAs comprising nucleotidesmismatched to the target can promote RNA directed Cas (e.g. C2c1 orC2c3) binding to a target sequence with little or no target cleavage.(See, e.g., Dahlman, 2015, Nat Biotechnol. 33(11):1159-1161, doi:10.1038/nbt.3390, published online 5 Oct. 2015). In an aspect, theinvention provides methods and mutations for modulating binding of Cas(e.g. C2c1 or C2c3) proteins that comprise nuclease activity. In certainembodiments, on-target binding is increased. In certain embodiments,off-target binding is decreased. In certain embodiments, on-targetbinding is decreased. In certain embodiments, off-target binding isincreased. In certain embodiments, there is increased or decreasedspecificity of on-target binding vs. off-target binding. In certainembodiments, nuclease activity of guide RNA-Cas (e.g. C2c1 or C2c3)enzyme is also modulated.

RNA-DNA heteroduplex formation is important for cleavage activity andspecificity throughout the target region, not only the seed regionsequence closest to the PAM. Thus, truncated guide RNAs show reducedcleavage activity and specificity. In an aspect, the invention providesmethod and mutations for increasing activity and specificity of cleavageusing altered guide RNAs.

The invention also demonstrates that modifications of Cas (e.g. C2c1 orC2c3) nuclease specificity can be made in concert with modifications totargeting range. Cas (e.g. C2c1 or C2c3) mutants can be designed thathave increased target specificity as well as accommodating modificationsin PAM recognition, for example by choosing mutations that alter PAMspecificity and combining those mutations with nt-groove mutations thatincrease (or if desired, decrease) specificity for on-target sequencesvs. off-target sequences. In one such embodiment, a PI domain residue ismutated to accommodate recognition of a desired PAM sequence while oneor more nt-groove amino acids is mutated to alter target specificity.The Cas (e.g. C2c1 or C2c3) methods and modifications described hereincan be used to counter loss of specificity resulting from alteration ofPAM recognition, enhance gain of specificity resulting from alterationof PAM recognition, counter gain of specificity resulting fromalteration of PAM recognition, or enhance loss of specificity resultingfrom alteration of PAM recognition.

The methods and mutations can be used with any Cas (e.g. C2c1 or C2c3)enzyme with altered PAM recognition. Non-limiting examples of PAMsincluded are as described herein elsewhere.

In further embodiments, the methods and mutations are used modifiedproteins.

In any of the non-naturally-occurring CRISPR enzymes, the CRISPR enzymemay comprise one or more heterologous functional domains.

The one or more heterologous functional domains may comprise one or morenuclear localization signal (NLS) domains. The one or more heterologousfunctional domains may comprise at least two or more NLSs.

The one or more heterologous functional domains may comprise one or moretranscriptional activation domains. A transcriptional activation domainmay comprise VP64.

The one or more heterologous functional domains may comprise one or moretranscriptional repression domains. A transcriptional repression domainmay comprise a KRAB domain or a SID domain.

The one or more heterologous functional domain may comprise one or morenuclease domains. The one or more nuclease domains may comprise Fok1.

The one or more heterologous functional domains may have one or more ofthe following activities: methylase activity, demethylase activity,transcription activation activity, transcription repression activity,transcription release factor activity, histone modification activity,nuclease activity, single-strand RNA cleavage activity, double-strandRNA cleavage activity, single-strand DNA cleavage activity,double-strand DNA cleavage activity and nucleic acid binding activity.

The at least one or more heterologous functional domains may be at ornear the amino-terminus of the enzyme and/or at or near thecarboxy-terminus of the enzyme.

The one or more heterologous functional domains may be fused to theCRISPR enzyme, or tethered to the CRISPR enzyme, or linked to the CRISPRenzyme by a linker moiety.

In any of the non-naturally-occurring CRISPR enzymes, the CRISPR enzymemay comprise a CRISPR enzyme from an organism from a genus comprisingAlicyclobacillus, Desulfovibrio, Desulfonatronum, Opitutaceae,Tuberibacillus, Bacillus, Brevibacillus, Desulfatirhabdium, Citrobacter,and Methylobacterium.

In any of the non-naturally-occurring CRISPR enzymes, the CRISPR enzymemay comprise a chimeric Cas (e.g. C2c1 or C2c3) enzyme comprising afirst fragment from a first Cas (e.g. C2c1 or C2c3) ortholog and asecond fragment from a second Cas (e.g. C2c1 or C2c3) ortholog, and thefirst and second Cas (e.g. C2c1 or C2c3) orthologs are different. Atleast one of the first and second Cas (e.g. C2c1 or C2c3) orthologs maycomprise a Cas (e.g. C2c1 or C2c3) from a species comprisingAlicyclobacillus acidoterrestris (e.g., ATCC 49025), Alicyclobacilluscontaminans (e.g., DSM 17975), Desulfovibrio inopinatus (e.g., DSM10711), Desulfonatronum thiodismutans (e.g., strain MLF-1), Opitutaceaebacterium TAV5, Tuberibacillus calidus (e.g., DSM 17572), Bacillusthermoamylovorans (e.g., strain B4166), Brevibacillus sp. CF 112,Bacillus sp. NSP2.1, Desulfatirhabdium butyrativorans (e.g., DSM 18734),Alicyclobacillus herbarius (e.g., DSM 13609), Citrobacter freundii(e.g., ATCC 8090), Brevibacillus agri (e.g., BAB-2500), andMethylobacterium nodulans (e.g., ORS 2060).

In any of the non-naturally-occurring CRISPR enzymes, a nucleotidesequence encoding the CRISPR enzyme may be codon optimized forexpression in a eukaryote.

In any of the non-naturally-occurring CRISPR enzymes, the cell may be aeukaryotic cell or a prokaryotic cell; wherein the CRISPR complex isoperable in the cell, and whereby the enzyme of the CRISPR complex hasreduced capability of modifying one or more off-target loci of the cellas compared to an unmodified enzyme and/or whereby the enzyme in theCRISPR complex has increased capability of modifying the one or moretarget loci as compared to an unmodified enzyme.

Accordingly, in an aspect, the invention provides a eukaryotic cellcomprising the engineered CRISPR protein or the system as definedherein.

In certain embodiments, the methods as described herein may compriseproviding a Cas (e.g. C2c1 or C2c3) transgenic cell in which one or morenucleic acids encoding one or more guide RNAs are provided or introducedoperably connected in the cell with a regulatory element comprising apromoter of one or more gene of interest. As used herein, the term “Castransgenic cell” refers to a cell, such as a eukaryotic cell, in which aCas gene has been genomically integrated. The nature, type, or origin ofthe cell are not particularly limiting according to the presentinvention. Also the way how the Cas transgene is introduced in the cellis may vary and can be any method as is known in the art. In certainembodiments, the Cas transgenic cell is obtained by introducing the Castransgene in an isolated cell. In certain other embodiments, the Castransgenic cell is obtained by isolating cells from a Cas transgenicorganism. By means of example, and without limitation, the Castransgenic cell as referred to herein may be derived from a Castransgenic eukaryote, such as a Cas knock-in eukaryote. Reference ismade to WO 2014/093622 (PCT/US13/74667), incorporated herein byreference. Methods of US Patent Publication Nos. 20120017290 and20110265198 assigned to Sangamo BioSciences, Inc. directed to targetingthe Rosa locus may be modified to utilize the CRISPR Cas system of thepresent invention. Methods of US Patent Publication No. 20130236946assigned to Cellectis directed to targeting the Rosa locus may also bemodified to utilize the CRISPR Cas system of the present invention. Bymeans of further example reference is made to Platt et. al. (Cell;159(2):440-455 (2014)), describing a Cas9 knock-in mouse, which isincorporated herein by reference, and which can be extrapolated to theCRISPR enzymes of the present invention as defined herein. The Castransgene can further comprise a Lox-Stop-polyA-Lox (LSL) cassettethereby rendering Cas expression inducible by Cre recombinase.Alternatively, the Cas transgenic cell may be obtained by introducingthe Cas transgene in an isolated cell. Delivery systems for transgenesare well known in the art. By means of example, the Cas transgene may bedelivered in for instance eukaryotic cell by means of vector (e.g., AAV,adenovirus, lentivirus) and/or particle and/or nanoparticle delivery, asalso described herein elsewhere.

It will be understood by the skilled person that the cell, such as theCas transgenic cell, as referred to herein may comprise further genomicalterations besides having an integrated Cas gene or the mutationsarising from the sequence specific action of Cas when complexed with RNAcapable of guiding Cas to a target locus, such as for instance one ormore oncogenic mutations, as for instance and without limitationdescribed in Platt et al. (2014), Chen et al., (2014) or Kumar et al..(2009).

The invention also provides a composition comprising the engineeredCRISPR protein as described herein, such as described in this section.

The invention also provides a non-naturally-occurring, engineeredcomposition comprising a CRISPR-Cas complex comprising any thenon-naturally-occurring CRISPR enzyme described above.

In an aspect, the invention provides in a vector system comprising oneor more vectors, wherein the one or more vectors comprises:

a) a first regulatory element operably linked to a nucleotide sequenceencoding the engineered CRISPR protein as defined herein; and optionally

b) a second regulatory element operably linked to one or more nucleotidesequences encoding one or more nucleic acid molecules comprising a guideRNA comprising a guide sequence, a direct repeat sequence, optionallywherein components (a) and (b) are located on same or different vectors.

The invention also provides a non-naturally-occurring, engineeredcomposition comprising:

a delivery system operably configured to deliver CRISPR-Cas complexcomponents or one or more polynucleotide sequences comprising orencoding said components into a cell, and wherein said CRISPR-Cascomplex is operable in the cell,

CRISPR-Cas complex components or one or more polynucleotide sequencesencoding for transcription and/or translation in the cell the CRISPR-Cascomplex components, comprising:

-   -   (I) the non-naturally-occurring CRISPR enzyme (e.g. engineered        C2c1 or C2c3) as described herein;    -   (II) CRISPR-Cas guide RNA comprising:    -   the guide sequence, and    -   a direct repeat sequence,    -   wherein the enzyme in the CRISPR complex has reduced capability        of modifying one or more off-target loci as compared to an        unmodified enzyme and/or whereby the enzyme in the CRISPR        complex has increased capability of modifying the one or more        target loci as compared to an unmodified enzyme.

In an aspect, the invention also provides in a system comprising theengineered CRISPR protein as described herein, such as described in thissection.

In any such compositions, the delivery system may comprise a yeastsystem, a lipofection system, a microinjection system, a biolisticsystem, virosomes, liposomes, immunoliposomes, polycations,lipid:nucleic acid conjugates or artificial virions, as defined hereinelsewhere.

In any such compositions, the delivery system may comprise a vectorsystem comprising one or more vectors, and wherein component (II)comprises a first regulatory element operably linked to a polynucleotidesequence which comprises the guide sequence, the direct repeat sequenceand optionally, and wherein component (I) comprises a second regulatoryelement operably linked to a polynucleotide sequence encoding the CRISPRenzyme.

In any such compositions, the delivery system may comprise a vectorsystem comprising one or more vectors, and wherein component (II)comprises a first regulatory element operably linked to the guidesequence and the direct repeat sequence, and wherein component (I)comprises a second regulatory element operably linked to apolynucleotide sequence encoding the CRISPR enzyme.

In any such compositions, the composition may comprise more than oneguide RNA, and each guide RNA has a different target whereby there ismultiplexing.

In any such compositions, the polynucleotide sequence(s) may be on onevector.

The invention also provides an engineered, non-naturally occurringClustered Regularly Interspersed Short Palindromic Repeats(CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) vector system comprisingone or more vectors comprising:

a) a first regulatory element operably linked to a nucleotide sequenceencoding a non-naturally-occurring CRISPR enzyme of any one of theinventive constructs herein; and

b) a second regulatory element operably linked to one or more nucleotidesequences encoding one or more of the guide RNAs, the guide RNAcomprising a guide sequence, a direct repeat sequence,

wherein:

components (a) and (b) are located on same or different vectors,

-   -   the CRISPR complex is formed;    -   the guide RNA targets the target polynucleotide loci and the        enzyme alters the polynucleotide loci, and    -   the enzyme in the CRISPR complex has reduced capability of        modifying one or more off-target loci as compared to an        unmodified enzyme and/or whereby the enzyme in the CRISPR        complex has increased capability of modifying the one or more        target loci as compared to an unmodified enzyme.

In such a system, component (II) may comprise a first regulatory elementoperably linked to a polynucleotide sequence which comprises the guidesequence, the direct repeat sequence, and wherein component (II) maycomprise a second regulatory element operably linked to a polynucleotidesequence encoding the CRISPR enzyme. In such a system, where applicablethe guide RNA may comprise a chimeric RNA.

In such a system, component (I) may comprise a first regulatory elementoperably linked to the guide sequence and the direct repeat sequence,and wherein component (II) may comprise a second regulatory elementoperably linked to a polynucleotide sequence encoding the CRISPR enzyme.Such a system may comprise more than one guide RNA, and each guide RNAhas a different target whereby there is multiplexing. Components (a) and(b) may be on the same vector.

In any such systems comprising vectors, the one or more vectors maycomprise one or more viral vectors, such as one or more retrovirus,lentivirus, adenovirus, adeno-associated virus or herpes simplex virus.

In any such systems comprising regulatory elements, at least one of saidregulatory elements may comprise a tissue-specific promoter. Thetissue-specific promoter may direct expression in a mammalian bloodcell, in a mammalian liver cell or in a mammalian eye.

In any of the above-described compositions or systems the direct repeatsequence, may comprise one or more protein-interacting RNA aptamers. Theone or more aptamers may be located in the tetraloop. The one or moreaptamers may be capable of binding MS2 bacteriophage coat protein.

In any of the above-described compositions or systems the cell may aeukaryotic cell or a prokaryotic cell; wherein the CRISPR complex isoperable in the cell, and whereby the enzyme of the CRISPR complex hasreduced capability of modifying one or more off-target loci of the cellas compared to an unmodified enzyme and/or whereby the enzyme in theCRISPR complex has increased capability of modifying the one or moretarget loci as compared to an unmodified enzyme.

The invention also provides a CRISPR complex of any of theabove-described compositions or from any of the above-described systems.

The invention also provides a method of modifying a locus of interest ina cell comprising contacting the cell with any of the herein-describedengineered CRISPR enzymes (e.g. engineered C2c1 or C2c3), compositionsor any of the herein-described systems or vector systems, or wherein thecell comprises any of the herein-described CRISPR complexes presentwithin the cell. In such methods the cell may be a prokaryotic oreukaryotic cell, preferably a eukaryotic cell. In such methods, anorganism may comprise the cell. In such methods the organism may not bea human or other animal.

Any such method may be ex vivo or in vitro.

In certain embodiments, a nucleotide sequence encoding at least one ofsaid guide RNA or Cas protein is operably connected in the cell with aregulatory element comprising a promoter of a gene of interest, wherebyexpression of at least one CRISPR-Cas system component is driven by thepromoter of the gene of interest. “Operably connected” is intended tomean that the nucleotide sequence encoding the guide RNA and/or the Casis linked to the regulatory element(s) in a manner that allows forexpression of the nucleotide sequence, as also referred to hereinelsewhere. The term “regulatory element” is also described hereinelsewhere. According to the invention, the regulatory element comprisesa promoter of a gene of interest, such as preferably a promoter of anendogenous gene of interest. In certain embodiments, the promoter is atits endogenous genomic location. In such embodiments, the nucleic acidencoding the CRISPR and/or Cas is under transcriptional control of thepromoter of the gene of interest at its native genomic location. Incertain other embodiments, the promoter is provided on a (separate)nucleic acid molecule, such as a vector or plasmid, or otherextrachromosomal nucleic acid, i.e. the promoter is not provided at itsnative genomic location. In certain embodiments, the promoter isgenomically integrated at a non-native genomic location.

Any such method, said modifying may comprise modulating gene expression.Said modulating gene expression may comprise activating gene expressionand/or repressing gene expression. Accordingly, in an aspect, theinvention provides in a method of modulating gene expression, whereinthe method comprises introducing the engineered CRISPR protein or systemas described herein into a cell.

The invention also provides a method of treating a disease, disorder orinfection in an individual in need thereof comprising administering aneffective amount of any of the engineered CRISPR enzymes (e.g.engineered C2c1 or C2c3), compositions, systems or CRISPR complexesdescribed herein. The disease, disorder or infection may comprise aviral infection. The viral infection may be HBV.

The invention also provides the use of any of the engineered CRISPRenzymes (e.g. engineered C2c1 or C2c3), compositions, systems or CRISPRcomplexes described above for gene or genome editing.

The invention also provides a method of altering the expression of agenomic locus of interest in a mammalian cell comprising contacting thecell with the engineered CRISPR enzymes (e.g. engineered C2c1 or C2c3),compositions, systems or CRISPR complexes described herein and therebydelivering the CRISPR-Cas (vector) and allowing the CRISPR-Cas complexto form and bind to target, and determining if the expression of thegenomic locus has been altered, such as increased or decreasedexpression, or modification of a gene product.

The invention also provides any of the engineered CRISPR enzymes (e.g.engineered C2c1 or C2c3), compositions, systems or CRISPR complexesdescribed above for use as a therapeutic. The therapeutic may be forgene or genome editing, or gene therapy.

In certain embodiments the activity of engineered CRISPR enzymes (e.g.engineered C2c1 or C2c3) as described herein comprises genomic DNAcleavage, optionally resulting in decreased transcription of a gene.

In an aspect, the invention provides in an isolated cell having alteredexpression of a genomic locus from the method s as described herein,wherein the altered expression is in comparison with a cell that has notbeen subjected to the method of altering the expression of the genomiclocus. In a related aspect, the invention provides in a cell lineestablished from such cell.

In one aspect, the invention provides a method of modifying an organismor a non-human organism by manipulation of a target sequence in agenomic locus of interest of for instance an HSC (hematopoietic stemcell), e.g., wherein the genomic locus of interest is associated with amutation associated with an aberrant protein expression or with adisease condition or state, comprising:

-   -   delivering to an HSC, e.g., via contacting an HSC with a        particle containing, a non-naturally occurring or engineered        composition comprising:        -   I. a CRISPR-Cas system guide RNA (gRNA) polynucleotide            sequence, comprising:            -   (a) a guide sequence capable of hybridizing to a target                sequence in a HSC,            -   (b) a direct repeat sequence, and        -   II. a CRISPR enzyme, optionally comprising at least one or            more nuclear localization sequences,

wherein, the guide sequence directs sequence-specific binding of aCRISPR complex to the target sequence, and

wherein the CRISPR complex comprises the CRISPR enzyme complexed with(1) the guide sequence that is hybridized to the target sequence, and

the method may optionally include also delivering a HDR template, e.g.,via the particle contacting the HSC containing or contacting the HSCwith another particle containing, the HDR template wherein the HDRtemplate provides expression of a normal or less aberrant form of theprotein; wherein “normal” is as to wild type, and “aberrant” can be aprotein expression that gives rise to a condition or disease state, and

optionally the method may include isolating or obtaining HSC from theorganism or non-human organism, optionally expanding the HSC population,performing contacting of the particle(s) with the HSC to obtain amodified HSC population, optionally expanding the population of modifiedHSCs, and optionally administering modified HSCs to the organism ornon-human organism.

In one aspect, the invention provides a method of modifying an organismor a non-human organism by manipulation of a target sequence in agenomic locus of interest of for instance a HSC, e.g., wherein thegenomic locus of interest is associated with a mutation associated withan aberrant protein expression or with a disease condition or state,comprising: delivering to an HSC, e.g., via contacting an HSC with aparticle containing, a non-naturally occurring or engineered compositioncomprising: I. (a) a guide sequence capable of hybridizing to a targetsequence in a HSC, and (b) at least one or more direct repeat sequences,and II. a CRISPR enzyme optionally having one or more NLSs, and theguide sequence directs sequence-specific binding of a CRISPR complex tothe target sequence, and wherein the CRISPR complex comprises the CRISPRenzyme complexed with the guide sequence that is hybridized to thetarget sequence; and

the method may optionally include also delivering a HDR template, e.g.,via the particle contacting the HSC containing or contacting the HSCwith another particle containing, the HDR template wherein the HDRtemplate provides expression of a normal or less aberrant form of theprotein; wherein “normal” is as to wild type, and “aberrant” can be aprotein expression that gives rise to a condition or disease state; and

optionally the method may include isolating or obtaining HSC from theorganism or non-human organism, optionally expanding the HSC population,performing contacting of the particle(s) with the HSC to obtain amodified HSC population, optionally expanding the population of modifiedHSCs, and optionally administering modified HSCs to the organism ornon-human organism.

The delivery can be of one or more polynucleotides encoding any one ormore or all of the CRISPR-complex, advantageously linked to one or moreregulatory elements for in vivo expression, e.g. via particle(s),containing a vector containing the polynucleotide(s) operably linked tothe regulatory element(s). Any or all of the polynucleotide sequenceencoding a CRISPR enzyme, guide sequence, direct repeat sequence, may beRNA. It will be appreciated that where reference is made to apolynucleotide, which is RNA and is said to ‘comprise’ a feature such adirect repeat sequence, the RNA sequence includes the feature. Where thepolynucleotide is DNA and is said to comprise a feature such a directrepeat sequence, the DNA sequence is or can be transcribed into the RNAincluding the feature at issue. Where the feature is a protein, such asthe CRISPR enzyme, the DNA or RNA sequence referred to is, or can be,translated (and in the case of DNA transcribed first).

In certain embodiments the invention provides a method of modifying anorganism, e.g., mammal including human or a non-human mammal or organismby manipulation of a target sequence in a genomic locus of interest ofan HSC e.g., wherein the genomic locus of interest is associated with amutation associated with an aberrant protein expression or with adisease condition or state, comprising delivering, e.g., via contactingof a non-naturally occurring or engineered composition with the HSC,wherein the composition comprises one or more particles comprisingviral, plasmid or nucleic acid molecule vector(s) (e.g. RNA) operablyencoding a composition for expression thereof, wherein the compositioncomprises: (A) I. a first regulatory element operably linked to aCRISPR-Cas system RNA polynucleotide sequence, wherein thepolynucleotide sequence comprises (a) a guide sequence capable ofhybridizing to a target sequence in a eukaryotic cell, (b) a directrepeat sequence and II. a second regulatory element operably linked toan enzyme-coding sequence encoding a CRISPR enzyme comprising at leastone or more nuclear localization sequences (or optionally at least oneor more nuclear localization sequences as some embodiments can involveno NLS), wherein (a), (b) and (c) are arranged in a 5′ to 3′orientation, wherein components I and II are located on the same ordifferent vectors of the system, wherein when transcribed and the guidesequence directs sequence-specific binding of a CRISPR complex to thetarget sequence, and wherein the CRISPR complex comprises the CRISPRenzyme complexed with the guide sequence that is hybridized to thetarget sequence, or (B) a non-naturally occurring or engineeredcomposition comprising a vector system comprising one or more vectorscomprising I. a first regulatory element operably linked to (a) a guidesequence capable of hybridizing to a target sequence in a eukaryoticcell, and (b) at least one or more direct repeat sequences, II. a secondregulatory element operably linked to an enzyme-coding sequence encodinga CRISPR enzyme, and optionally, where applicable, wherein components I,and II are located on the same or different vectors of the system,wherein when transcribed and the guide sequence directssequence-specific binding of a CRISPR complex to the target sequence,and wherein the CRISPR complex comprises the CRISPR enzyme complexedwith the guide sequence that is hybridized to the target sequence; themethod may optionally include also delivering a HDR template, e.g., viathe particle contacting the HSC containing or contacting the HSC withanother particle containing, the HDR template wherein the HDR templateprovides expression of a normal or less aberrant form of the protein;wherein “normal” is as to wild type, and “aberrant” can be a proteinexpression that gives rise to a condition or disease state; andoptionally the method may include isolating or obtaining HSC from theorganism or non-human organism, optionally expanding the HSC population,performing contacting of the particle(s) with the HSC to obtain amodified HSC population, optionally expanding the population of modifiedHSCs, and optionally administering modified HSCs to the organism ornon-human organism. In some embodiments, components I, II and III arelocated on the same vector. In other embodiments, components I and IIare located on the same vector, while component III is located onanother vector. In other embodiments, components I and III are locatedon the same vector, while component II is located on another vector. Inother embodiments, components II and III are located on the same vector,while component I is located on another vector. In other embodiments,each of components I, II and III is located on different vectors. Theinvention also provides a viral or plasmid vector system as describedherein.

By manipulation of a target sequence, Applicants also mean theepigenetic manipulation of a target sequence. This may be of thechromatin state of a target sequence, such as by modification of themethylation state of the target sequence (i.e. addition or removal ofmethylation or methylation patterns or CpG islands), histonemodification, increasing or reducing accessibility to the targetsequence, or by promoting 3D folding. It will be appreciated that wherereference is made to a method of modifying an organism or mammalincluding human or a non-human mammal or organism by manipulation of atarget sequence in a genomic locus of interest, this may apply to theorganism (or mammal) as a whole or just a single cell or population ofcells from that organism (if the organism is multicellular). In the caseof humans, for instance, Applicants envisage, inter alia, a single cellor a population of cells and these may preferably be modified ex vivoand then re-introduced. In this case, a biopsy or other tissue orbiological fluid sample may be necessary. Stem cells are alsoparticularly preferred in this regard. But, of course, in vivoembodiments are also envisaged. And the invention is especiallyadvantageous as to HSCs.

The invention in some embodiments comprehends a method of modifying anorganism or a non-human organism by manipulation of a first and a secondtarget sequence on opposite strands of a DNA duplex in a genomic locusof interest in a HSC e.g., wherein the genomic locus of interest isassociated with a mutation associated with an aberrant proteinexpression or with a disease condition or state, comprising delivering,e.g., by contacting HSCs with particle(s) comprising a non-naturallyoccurring or engineered composition comprising:

-   -   I. a first CRISPR-Cas (e.g. C2c1 or C2c3) system RNA        polynucleotide sequence, wherein the first polynucleotide        sequence comprises:        -   (a) a first guide sequence capable of hybridizing to the            first target sequence,        -   (b) a first direct repeat sequence, and    -   II. a second CRISPR-Cas (e.g. C2c1 or C2c3) system guide RNA        polynucleotide sequence, wherein the second polynucleotide        sequence comprises:        -   (a) a second guide sequence capable of hybridizing to the            second target sequence,        -   (b) a second direct repeat sequence, and    -   III. a polynucleotide sequence encoding a CRISPR enzyme        comprising at least one or more nuclear localization sequences        and comprising one or more mutations, wherein (a), (b) and (c)        are arranged in a 5′ to 3′ orientation; or    -   IV. expression product(s) of one or more of I. to III., e.g.,        the first and the second direct repeat sequence, the CRISPR        enzyme;

wherein when transcribed, the first and the second guide sequencedirects sequence-specific binding of a first and a second CRISPR complexto the first and second target sequences respectively, wherein the firstCRISPR complex comprises the CRISPR enzyme complexed with (1) the firstguide sequence that is hybridized to the first target sequence, whereinthe second CRISPR complex comprises the CRISPR enzyme complexed with (1)the second guide sequence that is hybridized to the second targetsequence, wherein the polynucleotide sequence encoding a CRISPR enzymeis DNA or RNA, and wherein the first guide sequence directs cleavage ofone strand of the DNA duplex near the first target sequence and thesecond guide sequence directs cleavage of the other strand near thesecond target sequence inducing a double strand break, thereby modifyingthe organism or the non-human organism; and the method may optionallyinclude also delivering a HDR template, e.g., via the particlecontacting the HSC containing or contacting the HSC with anotherparticle containing, the HDR template wherein the HDR template providesexpression of a normal or less aberrant form of the protein; wherein“normal” is as to wild type, and “aberrant” can be a protein expressionthat gives rise to a condition or disease state; and optionally themethod may include isolating or obtaining HSC from the organism ornon-human organism, optionally expanding the HSC population, performingcontacting of the particle(s) with the HSC to obtain a modified HSCpopulation, optionally expanding the population of modified HSCs, andoptionally administering modified HSCs to the organism or non-humanorganism. In some methods of the invention any or all of thepolynucleotide sequence encoding the CRISPR enzyme, the first and thesecond guide sequence, the first and the second direct repeat sequence.In further embodiments of the invention the polynucleotides encoding thesequence encoding the CRISPR enzyme, the first and the second guidesequence, the first and the second direct repeat sequence, is/are RNAand are delivered via liposomes, nanoparticles, exosomes, microvesicles,or a gene-gun; but, it is advantageous that the delivery is via aparticle. In certain embodiments of the invention, the first and seconddirect repeat sequence share 100% identity. In some embodiments, thepolynucleotides may be comprised within a vector system comprising oneor more vectors. In preferred embodiments, the first CRISPR enzyme hasone or more mutations such that the enzyme is a complementary strandnicking enzyme, and the second CRISPR enzyme has one or more mutationssuch that the enzyme is a non-complementary strand nicking enzyme.Alternatively the first enzyme may be a non-complementary strand nickingenzyme, and the second enzyme may be a complementary strand nickingenzyme. In preferred methods of the invention the first guide sequencedirecting cleavage of one strand of the DNA duplex near the first targetsequence and the second guide sequence directing cleavage of the otherstrand near the second target sequence results in a 5′ overhang. Inembodiments of the invention the 5′ overhang is at most 200 base pairs,preferably at most 100 base pairs, or more preferably at most 50 basepairs. In embodiments of the invention the 5′ overhang is at least 26base pairs, preferably at least 30 base pairs or more preferably 34-50base pairs.

The invention in some embodiments comprehends a method of modifying anorganism or a non-human organism by manipulation of a first and a secondtarget sequence on opposite strands of a DNA duplex in a genomic locusof interest in for instance a HSC e.g., wherein the genomic locus ofinterest is associated with a mutation associated with an aberrantprotein expression or with a disease condition or state, comprisingdelivering, e.g., by contacting HSCs with particle(s) comprising anon-naturally occurring or engineered composition comprising:

-   -   I. a first regulatory element operably linked to        -   (a) a first guide sequence capable of hybridizing to the            first target sequence, and        -   (b) at least one or more direct repeat sequences,    -   II. a second regulatory element operably linked to        -   (a) a second guide sequence capable of hybridizing to the            second target sequence, and        -   (b) at least one or more direct repeat sequences,    -   III. a third regulatory element operably linked to an        enzyme-coding sequence encoding a CRISPR enzyme (e.g. C2c1 or        C2c3), and    -   V. expression product(s) of one or more of I. to IV., e.g., the        first and the second direct repeat sequence, the CRISPR enzyme;        wherein components I, IL, III and IV are located on the same or        different vectors of the system, when transcribed, and the first        and the second guide sequence direct sequence-specific binding        of a first and a second CRISPR complex to the first and second        target sequences respectively, wherein the first CRISPR complex        comprises the CRISPR enzyme complexed with (1) the first guide        sequence that is hybridized to the first target sequence,        wherein the second CRISPR complex comprises the CRISPR enzyme        complexed with the second guide sequence that is hybridized to        the second target sequence, wherein the polynucleotide sequence        encoding a CRISPR enzyme is DNA or RNA, and wherein the first        guide sequence directs cleavage of one strand of the DNA duplex        near the first target sequence and the second guide sequence        directs cleavage of the other strand near the second target        sequence inducing a double strand break, thereby modifying the        organism or the non-human organism; and the method may        optionally include also delivering a HDR template, e.g., via the        particle contacting the HSC containing or contacting the HSC        with another particle containing, the HDR template wherein the        HDR template provides expression of a normal or less aberrant        form of the protein; wherein “normal” is as to wild type, and        “aberrant” can be a protein expression that gives rise to a        condition or disease state; and optionally the method may        include isolating or obtaining HSC from the organism or        non-human organism, optionally expanding the HSC population,        performing contacting of the particle(s) with the HSC to obtain        a modified HSC population, optionally expanding the population        of modified HSCs, and optionally administering modified HSCs to        the organism or non-human organism.

The invention also provides a vector system as described herein. Thesystem may comprise one, two, three or four different vectors.Components I, II, III and IV may thus be located on one, two, three orfour different vectors, and all combinations for possible locations ofthe components are herein envisaged, for example: components I, II, IIIand IV can be located on the same vector; components I, II, III and IVcan each be located on different vectors; components I, II, II I and IVmay be located on a total of two or three different vectors, with allcombinations of locations envisaged, etc. In some methods of theinvention any or all of the polynucleotide sequence encoding the CRISPRenzyme, the first and the second guide sequence, the first and thesecond direct repeat sequence is/are RNA. In further embodiments of theinvention the first and second direct repeat sequence share 100%identity. In preferred embodiments, the first CRISPR enzyme has one ormore mutations such that the enzyme is a complementary strand nickingenzyme, and the second CRISPR enzyme has one or more mutations such thatthe enzyme is a non-complementary strand nicking enzyme. Alternativelythe first enzyme may be a non-complementary strand nicking enzyme, andthe second enzyme may be a complementary strand nicking enzyme. In afurther embodiment of the invention, one or more of the viral vectorsare delivered via liposomes, nanoparticles, exosomes, microvesicles, ora gene-gun; but, particle delivery is advantageous.

In preferred methods of the invention the first guide sequence directingcleavage of one strand of the DNA duplex near the first target sequenceand the second guide sequence directing cleavage of other strand nearthe second target sequence results in a 5′ overhang. In embodiments ofthe invention the 5′ overhang is at most 200 base pairs, preferably atmost 100 base pairs, or more preferably at most 50 base pairs. Inembodiments of the invention the 5′ overhang is at least 26 base pairs,preferably at least 30 base pairs or more preferably 34-50 base pairs.

The invention in some embodiments comprehends a method of modifying agenomic locus of interest in for instance HSC e.g., wherein the genomiclocus of interest is associated with a mutation associated with anaberrant protein expression or with a disease condition or state, byintroducing into the HSC, e.g., by contacting HSCs with particle(s)comprising, a Cas protein having one or more mutations and two guideRNAs that target a first strand and a second strand of the DNA moleculerespectively in the HSC, whereby the guide RNAs target the DNA moleculeand the Cas protein nicks each of the first strand and the second strandof the DNA molecule, whereby a target in the HSC is altered; and,wherein the Cas protein and the two guide RNAs do not naturally occurtogether and the method may optionally include also delivering a HDRtemplate, e.g., via the particle contacting the HSC containing orcontacting the HSC with another particle containing, the HDR templatewherein the HDR template provides expression of a normal or lessaberrant form of the protein; wherein “normal” is as to wild type, and“aberrant” can be a protein expression that gives rise to a condition ordisease state; and optionally the method may include isolating orobtaining HSC from the organism or non-human organism, optionallyexpanding the HSC population, performing contacting of the particle(s)with the HSC to obtain a modified HSC population, optionally expandingthe population of modified HSCs, and optionally administering modifiedHSCs to the organism or non-human organism. In preferred methods of theinvention the Cas protein nicking each of the first strand and thesecond strand of the DNA molecule results in a 5′ overhang. Inembodiments of the invention the 5′ overhang is at most 200 base pairs,preferably at most 100 base pairs, or more preferably at most 50 basepairs. In embodiments of the invention the 5′ overhang is at least 26base pairs, preferably at least 30 base pairs or more preferably 34-50base pairs. In an aspect of the invention the Cas protein is codonoptimized for expression in a eukaryotic cell, preferably a mammaliancell or a human cell. Aspects of the invention relate to the expressionof a gene product being decreased or a template polynucleotide beingfurther introduced into the DNA molecule encoding the gene product or anintervening sequence being excised precisely by allowing the two 5′overhangs to reanneal and ligate or the activity or function of the geneproduct being altered or the expression of the gene product beingincreased. In an embodiment of the invention, the gene product is aprotein.

The invention in some embodiments comprehends a method of modifying agenomic locus of interest in for instance HSC e.g., wherein the genomiclocus of interest is associated with a mutation associated with anaberrant protein expression or with a disease condition or state, byintroducing into the HSC, e.g., by contacting HSCs with particle(s)comprising,

-   -   a) a first regulatory element operably linked to each of two        CRISPR-Cas system guide RNAs that target a first strand and a        second strand respectively of a double stranded DNA molecule of        the HSC, and    -   b) a second regulatory element operably linked to a Cas (e.g.        C2c1 or C2c3) protein, or    -   c) expression product(s) of a) or b),        wherein components (a) and (b) are located on same or different        vectors of the system, whereby the guide RNAs target the DNA        molecule of the HSC and the Cas protein nicks each of the first        strand and the second strand of the DNA molecule of the HSC;        and, wherein the Cas protein and the two guide RNAs do not        naturally occur together; and the method may optionally include        also delivering a HDR template, e.g., via the particle        contacting the HSC containing or contacting the HSC with another        particle containing, the HDR template wherein the HDR template        provides expression of a normal or less aberrant form of the        protein; wherein “normal” is as to wild type, and “aberrant” can        be a protein expression that gives rise to a condition or        disease state; and optionally the method may include isolating        or obtaining HSC from the organism or non-human organism,        optionally expanding the HSC population, performing contacting        of the particle(s) with the HSC to obtain a modified HSC        population, optionally expanding the population of modified        HSCs, and optionally administering modified HSCs to the organism        or non-human organism. In aspects of the invention the guide        RNAs may comprise a guide sequence fused to a direct repeat        sequence. Aspects of the invention relate to the expression of a        gene product being decreased or a template polynucleotide being        further introduced into the DNA molecule encoding the gene        product or an intervening sequence being excised precisely by        allowing the two 5′ overhangs to reanneal and ligate or the        activity or function of the gene product being altered or the        expression of the gene product being increased. In an embodiment        of the invention, the gene product is a protein. In preferred        embodiments of the invention the vectors of the system are viral        vectors. In a further embodiment, the vectors of the system are        delivered via liposomes, nanoparticles, exosomes, microvesicles,        or a gene-gun; and particles are preferred. In one aspect, the        invention provides a method of modifying a target polynucleotide        in a HSC. In some embodiments, the method comprises allowing a        CRISPR complex to bind to the target polynucleotide to effect        cleavage of said target polynucleotide thereby modifying the        target polynucleotide, wherein the CRISPR complex comprises a        CRISPR enzyme complexed with a guide sequence hybridized to a        target sequence within said target polynucleotide, wherein said        guide sequence is linked to a direct repeat sequence. In some        embodiments, said cleavage comprises cleaving one or two strands        at the location of the target sequence by said CRISPR enzyme. In        some embodiments, said cleavage results in decreased        transcription of a target gene. In some embodiments, the method        further comprises repairing said cleaved target polynucleotide        by homologous recombination with an exogenous template        polynucleotide, wherein said repair results in a mutation        comprising an insertion, deletion, or substitution of one or        more nucleotides of said target polynucleotide. In some        embodiments, said mutation results in one or more amino acid        changes in a protein expressed from a gene comprising the target        sequence. In some embodiments, the method further comprises        delivering one or more vectors or expression product(s) thereof,        e.g., via particle(s), to for instance said HSC, wherein the one        or more vectors drive expression of one or more of: the CRISPR        enzyme, the guide sequence linked to the direct repeat sequence.        In some embodiments, said vectors are delivered to for instance        the HSC in a subject. In some embodiments, said modifying takes        place in said HSC in a cell culture. In some embodiments, the        method further comprises isolating said HSC from a subject prior        to said modifying. In some embodiments, the method further        comprises returning said HSC and/or cells derived therefrom to        said subject.

In one aspect, the invention provides a method of generating forinstance a HSC comprising a mutated disease gene. In some embodiments, adisease gene is any gene associated with an increase in the risk ofhaving or developing a disease. In some embodiments, the methodcomprises (a) introducing one or more vectors or expression product(s)thereof, e.g., via particle(s), into a HSC, wherein the one or morevectors drive expression of one or more of: a CRISPR enzyme, a guidesequence linked to a direct repeat sequence; and (b) allowing a CRISPRcomplex to bind to a target polynucleotide to effect cleavage of thetarget polynucleotide within said disease gene, wherein the CRISPRcomplex comprises the CRISPR enzyme complexed with the guide sequencethat is hybridized to the target sequence within the targetpolynucleotide, and optionally, where applicable, thereby generating aHSC comprising a mutated disease gene. In some embodiments, saidcleavage comprises cleaving one or two strands at the location of thetarget sequence by said CRISPR enzyme. In some embodiments, saidcleavage results in decreased transcription of a target gene. In someembodiments, the method further comprises repairing said cleaved targetpolynucleotide by homologous recombination with an exogenous templatepolynucleotide, wherein said repair results in a mutation comprising aninsertion, deletion, or substitution of one or more nucleotides of saidtarget polynucleotide. In some embodiments, said mutation results in oneor more amino acid changes in a protein expression from a genecomprising the target sequence. In some embodiments the modified HSC isadministered to an animal to thereby generate an animal model.

In one aspect, the invention provides for methods of modifying a targetpolynucleotide in for instance a HSC. In some embodiments, the methodcomprises allowing a CRISPR complex to bind to the target polynucleotideto effect cleavage of said target polynucleotide thereby modifying thetarget polynucleotide, wherein the CRISPR complex comprises a CRISPRenzyme complexed with a guide sequence hybridized to a target sequencewithin said target polynucleotide, wherein said guide sequence is linkedto a direct repeat sequence. In other embodiments, this inventionprovides a method of modifying expression of a polynucleotide in aeukaryotic cell that arises from for instance an HSC. The methodcomprises increasing or decreasing expression of a target polynucleotideby using a CRISPR complex that binds to the polynucleotide in the HSC;advantageously the CRISPR complex is delivered via particle(s).

In some methods, a target polynucleotide can be inactivated to effectthe modification of the expression in for instance an HSC. For example,upon the binding of a CRISPR complex to a target sequence in a cell, thetarget polynucleotide is inactivated such that the sequence is nottranscribed, the coded protein is not produced, or the sequence does notfunction as the wild-type sequence does.

In some embodiments the RNA of the CRISPR-Cas system, e.g., the guide orgRNA, can be modified; for instance to include an aptamer or afunctional domain. An aptamer is a synthetic oligonucleotide that bindsto a specific target molecule; for instance a nucleic acid molecule thathas been engineered through repeated rounds of in vitro selection orSELEX (systematic evolution of ligands by exponential enrichment) tobind to various molecular targets such as small molecules, proteins,nucleic acids, and even cells, tissues and organisms. Aptamers areuseful in that they offer molecular recognition properties that rivalthat of antibodies. In addition to their discriminate recognition,aptamers offer advantages over antibodies including that they elicitlittle or no immunogenicity in therapeutic applications. Accordingly, inthe practice of the invention, either or both of the enzyme or the RNAcan include a functional domain.

In some embodiments, the functional domain is a transcriptionalactivation domain, preferably VP64. In some embodiments, the functionaldomain is a transcription repression domain, preferably KRAB. In someembodiments, the transcription repression domain is SID, or concatemersof SID (eg SID4X). In some embodiments, the functional domain is anepigenetic modifying domain, such that an epigenetic modifying enzyme isprovided. In some embodiments, the functional domain is an activationdomain, which may be the P65 activation domain. In some embodiments, thefunctional domain comprises nuclease activity. In one such embodiment,the functional domain comprises Fok1.

The invention also provides an in vitro or ex vivo cell comprising anyof the modified CRISPR enzymes, compositions, systems or complexesdescribed above, or from any of the methods described above. The cellmay be a eukaryotic cell or a prokaryotic cell. The invention alsoprovides progeny of such cells. The invention also provides a product ofany such cell or of any such progeny, wherein the product is a productof the said one or more target loci as modified by the modified CRISPRenzyme of the CRISPR complex. The product may be a peptide, polypeptideor protein. Some such products may be modified by the modified CRISPRenzyme of the CRISPR complex. In some such modified products, theproduct of the target locus is physically distinct from the product ofthe said target locus which has not been modified by the said modifiedCRISPR enzyme.

The invention also provides a polynucleotide molecule comprising apolynucleotide sequence encoding any of the non-naturally-occurringCRISPR enzymes described above.

Any such polynucleotide may further comprise one or more regulatoryelements which are operably linked to the polynucleotide sequenceencoding the non-naturally-occurring CRISPR enzyme.

In any such polynucleotide which comprises one or more regulatoryelements, the one or more regulatory elements may be operably configuredfor expression of the non-naturally-occurring CRISPR enzyme in aeukaryotic cell. The eukaryotic cell may be a human cell. The eukaryoticcell may be a rodent cell, optionally a mouse cell. The eukaryotic cellmay be a yeast cell. The eukaryotic cell may be a chinese hamster ovary(CHO) cell. The eukaryotic cell may be an insect cell.

In any such polynucleotide which comprises one or more regulatoryelements, the one or more regulatory elements may be operably configuredfor expression of the non-naturally-occurring CRISPR enzyme in aprokaryotic cell.

In any such polynucleotide which comprises one or more regulatoryelements, the one or more regulatory elements may operably configuredfor expression of the non-naturally-occurring CRISPR enzyme in an invitro system.

The invention also provides an expression vector comprising any of theabove-described polynucleotide molecules. The invention also providessuch polynucleotide molecule(s), for instance such polynucleotidemolecules operably configured to express the protein and/or the nucleicacid component(s), as well as such vector(s).

The invention further provides for a method of making mutations to a Cas(e.g. C2c1 or C2c3) or a mutated or modified Cas (e.g. C2c1 or C2c3)that is an ortholog of the CRISPR enzymes according to the invention asdescribed herein, comprising ascertaining amino acid(s) in that orthologmay be in close proximity or may touch a nucleic acid molecule, e.g.,DNA, RNA, gRNA, etc., and/or amino acid(s) analogous or corresponding toherein-identified amino acid(s) in CRISPR enzymes according to theinvention as described herein for modification and/or mutation, andsynthesizing or preparing or expressing the orthologue comprising,consisting of or consisting essentially of modification(s) and/ormutation(s) or mutating as herein-discussed, e.g., modifying, e.g.,changing or mutating, a neutral amino acid to a charged, e.g.,positively charged, amino acid, e.g., Alanine. The so modified orthologcan be used in CRISPR-Cas systems; and nucleic acid molecule(s)expressing it may be used in vector or other delivery systems thatdeliver molecules or encoding CRISPR-Cas system components asherein-discussed.

In an aspect, the invention provides efficient on-target activity andminimizes off target activity. In an aspect, the invention providesefficient on-target cleavage by a CRISPR protein and minimizesoff-target cleavage by the CRISPR protein. In an aspect, the inventionprovides guide specific binding of a CRISPR protein at a gene locuswithout DNA cleavage. In an aspect, the invention provides efficientguide directed on-target binding of a CRISPR protein at a gene locus andminimizes off-target binding of the CRISPR protein. Accordingly, in anaspect, the invention provides target-specific gene regulation. In anaspect, the invention provides guide specific binding of a CRISPR enzymeat a gene locus without DNA cleavage. Accordingly, in an aspect, theinvention provides for cleavage at one gene locus and gene regulation ata different gene locus using a single CRISPR enzyme. In an aspect, theinvention provides orthogonal activation and/or inhibition and/orcleavage of multiple targets using one or more CRISPR protein and/orenzyme.

In another aspect, the present invention provides for a method offunctional screening of genes in a genome in a pool of cells ex vivo orin vivo comprising the administration or expression of a librarycomprising a plurality of CRISPR-Cas system guide RNAs (gRNAs) andwherein the screening further comprises use of a CRISPR enzyme, whereinthe CRISPR complex is modified to comprise a heterologous functionaldomain. In an aspect the invention provides a method for screening agenome comprising the administration to a host or expression in a hostin vivo of a library. In an aspect the invention provides a method asherein discussed further comprising an activator administered to thehost or expressed in the host. In an aspect the invention provides amethod as herein discussed wherein the activator is attached to a CRISPRprotein. In an aspect the invention provides a method as hereindiscussed wherein the activator is attached to the N terminus or the Cterminus of the CRISPR protein. In an aspect the invention provides amethod as herein discussed wherein the activator is attached to a gRNAloop. In an aspect the invention provides a method as herein discussedfurther comprising a repressor administered to the host or expressed inthe host. In an aspect the invention provides a method as hereindiscussed wherein the screening comprises affecting and detecting geneactivation, gene inhibition, or cleavage in the locus.

In an aspect the invention provides a method as herein discussed whereinthe host is a eukaryotic cell. In an aspect the invention provides amethod as herein discussed wherein the host is a mammalian cell. In anaspect the invention provides a method as herein discussed, wherein thehost is a non-human eukaryote cell. In an aspect the invention providesa method as herein discussed, wherein the non-human eukaryote cell is anon-human mammal cell. In an aspect the invention provides a method asherein discussed, wherein the non-human mammal cell may be including,but not limited to, primate bovine, ovine, porcine, canine, rodent,Leporidae such as monkey, cow, sheep, pig, dog, rabbit, rat or mousecell. In an aspect the invention provides a method as herein discussed,the cell may be a non-mammalian eukaryotic cell such as poultry bird(e.g., chicken), vertebrate fish (e.g., salmon) or shellfish (e.g.,oyster, claim, lobster, shrimp) cell. In an aspect the inventionprovides a method as herein discussed, the non-human eukaryote cell is aplant cell. The plant cell may be of a monocot or dicot or of a crop orgrain plant such as cassava, com, sorghum, soybean, wheat, oat or rice.The plant cell may also be of an algae, tree or production plant, fruitor vegetable (e.g., trees such as citrus trees, e.g., orange, grapefruitor lemon trees; peach or nectarine trees; apple or pear trees; nut treessuch as almond or walnut or pistachio trees; nightshade plants; plantsof the genus Brassica; plants of the genus Lactuca; plants of the genusSpinacia; plants of the genus Capsicum; cotton, tobacco, asparagus,carrot, cabbage, broccoli, cauliflower, tomato, eggplant, pepper,lettuce, spinach, strawberry, blueberry, raspberry, blackberry, grape,coffee, cocoa, etc).

In an aspect the invention provides a method as herein discussedcomprising the delivery of the CRISPR-Cas complexes or component(s)thereof or nucleic acid molecule(s) coding therefor, wherein saidnucleic acid molecule(s) are operatively linked to regulatorysequence(s) and expressed in vivo. In an aspect the invention provides amethod as herein discussed wherein the expressing in vivo is via alentivirus, an adenovirus, or an AAV. In an aspect the inventionprovides a method as herein discussed wherein the delivery is via aparticle, a nanoparticle, a lipid or a cell penetrating peptide (CPP).

In particular embodiments it can be of interest to target the CRISPR-Cascomplex to the chloroplast. In many cases, this targeting may beachieved by the presence of an N-terminal extension, called achloroplast transit peptide (CTP) or plastid transit peptide.Chromosomal transgenes from bacterial sources must have a sequenceencoding a CTP sequence fused to a sequence encoding an expressedpolypeptide if the expressed polypeptide is to be compartmentalized inthe plant plastid (e.g. chloroplast). Accordingly, localization of anexogenous polypeptide to a chloroplast is often 1 accomplished by meansof operably linking a polynucleotide sequence encoding a CTP sequence tothe 5′ region of a polynucleotide encoding the exogenous polypeptide.The CTP is removed in a processing step during translocation into theplastid. Processing efficiency may, however, be affected by the aminoacid sequence of the CTP and nearby sequences at the amino (NH₂)terminus of the peptide. Other options for targeting to the chloroplastwhich have been described are the maize cab-m7 signal sequence (U.S.Pat. No. 7,022,896, WO 97/41228) a pea glutathione reductase signalsequence (WO 97/41228) and the CTP described in US2009029861.

In an aspect the invention provides a pair of CRISPR-Cas complexes, eachcomprising a guide RNA (gRNA) comprising a guide sequence capable ofhybridizing to a target sequence in a genomic locus of interest in acell, wherein at least one loop of each sgRNA is modified by theinsertion of distinct RNA sequence(s) that bind to one or more adaptorproteins, and wherein the adaptor protein is associated with one or morefunctional domains, wherein each gRNA of each CRISPR-Cas comprises afunctional domain having a DNA cleavage activity. In an aspect theinvention provides a paired CRISPR-Cas complexes as herein-discussed,wherein the DNA cleavage activity is due to a Fok1 nuclease.

In an aspect the invention provides a method for cutting a targetsequence in a genomic locus of interest comprising delivery to a cell ofthe CRISPR-Cas complexes or component(s) thereof or nucleic acidmolecule(s) coding therefor, wherein said nucleic acid molecule(s) areoperatively linked to regulatory sequence(s) and expressed in vivo. Inan aspect the invention provides a method as herein-discussed whereinthe delivery is via a lentivirus, an adenovirus, or an AAV. In an aspectthe invention provides a method as herein-discussed or paired CRISPR-Cascomplexes as herein-discussed wherein the target sequence for a firstcomplex of the pair is on a first strand of double stranded DNA and thetarget sequence for a second complex of the pair is on a second strandof double stranded DNA. In an aspect the invention provides a method asherein-discussed or paired CRISPR-Cas complexes as herein-discussedwherein the target sequences of the first and second complexes are inproximity to each other such that the DNA is cut in a manner thatfacilitates homology directed repair. In an aspect a herein method canfurther include introducing into the cell template DNA. In an aspect aherein method or herein paired CRISPR-Cas complexes can involve whereineach CRISPR-Cas complex has a CRISPR enzyme that is mutated such that ithas no more than about 5% of the nuclease activity of the CRISPR enzymethat is not mutated.

In an aspect the invention provides a library, method or complex asherein-discussed wherein the gRNA is modified to have at least onenon-coding functional loop, e.g., wherein the at least one non-codingfunctional loop is repressive; for instance, wherein the at least onenon-coding functional loop comprises Alu.

In one aspect, the invention provides a method for altering or modifyingexpression of a gene product. The said method may comprise introducinginto a cell containing and expressing a DNA molecule encoding the geneproduct an engineered, non-naturally occurring CRISPR-Cas systemcomprising a Cas protein and guide RNA that targets the DNA molecule,whereby the guide RNA targets the DNA molecule encoding the gene productand the Cas protein cleaves the DNA molecule encoding the gene product,whereby expression of the gene product is altered; and, wherein the Casprotein and the guide RNA do not naturally occur together. The inventionfurther comprehends the Cas protein being codon optimized for expressionin a Eukaryotic cell. In a preferred embodiment the Eukaryotic cell is amammalian cell and in a more preferred embodiment the mammalian cell isa human cell. In a further embodiment of the invention, the expressionof the gene product is decreased.

In an aspect, the invention provides altered cells and progeny of thosecells, as well as products made by the cells. CRISPR-Cas (e.g. C2c1)proteins and systems of the invention are used to produce cellscomprising a modified target locus. In some embodiments, the method maycomprise allowing a nucleic acid-targeting complex to bind to the targetDNA or RNA to effect cleavage of said target DNA or RNA therebymodifying the target DNA or RNA, wherein the nucleic acid-targetingcomplex comprises a nucleic acid-targeting effector protein complexedwith a guide RNA hybridized to a target sequence within said target DNAor RNA. In one aspect, the invention provides a method of repairing agenetic locus in a cell. In another aspect, the invention provides amethod of modifying expression of DNA or RNA in a eukaryotic cell. Insome embodiments, the method comprises allowing a nucleic acid-targetingcomplex to bind to the DNA or RNA such that said binding results inincreased or decreased expression of said DNA or RNA; wherein thenucleic acid-targeting complex comprises a nucleic acid-targetingeffector protein complexed with a guide RNA. Similar considerations andconditions apply as above for methods of modifying a target DNA or RNA.In fact, these sampling, culturing and re-introduction options applyacross the aspects of the present invention. In an aspect, the inventionprovides for methods of modifying a target DNA or RNA in a eukaryoticcell, which may be in vivo, ex vivo or in vitro. In some embodiments,the method comprises sampling a cell or population of cells from a humanor non-human animal, and modifying the cell or cells. Culturing mayoccur at any stage ex vivo. Such cells can be, without limitation, plantcells, animal cells, particular cell types of any organism, includingstem cells, immune cells, T cell, B cells, dendritic cells,cardiovascular cells, epithelial cells, stem cells and the like. Thecells can be modified according to the invention to produce geneproducts, for example in controlled amounts, which may be increased ordecreased, depending on use, and/or mutated. In certain embodiments, agenetic locus of the cell is repaired. The cell or cells may even bere-introduced into the non-human animal or plant. For re-introducedcells it may be preferred that the cells are stem cells.

In an aspect, the invention provides cells which transiently compriseCRISPR systems, or components. For example, CRISPR proteins or enzymesand nucleic acids are transiently provided to a cell and a genetic locusis altered, followed by a decline in the amount of one or morecomponents of the CRISPR system. Subsequently, the cells, progeny of thecells, and organisms which comprise the cells, having acquired a CRISPRmediated genetic alteration, comprise a diminished amount of one or moreCRISPR system components, or no longer contain the one or more CRISPRsystem components. One non-limiting example is a self-inactivatingCRISPR-Cas system such as further described herein. Thus, the inventionprovides cells, and organisms, and progeny of the cells and organismswhich comprise one or more CRISPR-Cas system-altered genetic loci, butessentially lack one or more CRISPR system component. In certainembodiments, the CRISPR system components are substantially absent. Suchcells, tissues and organisms advantageously comprise a desired orselected genetic alteration but have lost CRISPR-Cas components orremnants thereof that potentially might act non-specifically, lead toquestions of safety, or hinder regulatory approval. As well, theinvention provides products made by the cells, organisms, and progeny ofthe cells and organisms.

Inducible C2c1 or Inducible C2c3 CRISPR-Cas Systems (“Split-C2c1” or“Split-C2c3”)

In an aspect the invention provides a non-naturally occurring orengineered inducible C2c1 or inducible C2c3 CRISPR-Cas system,comprising:

a first C2c1 or C2c3 fusion construct attached to a first half of aninducible dimer and

a second C2c1 or C2c3 fusion construct attached to a second half of theinducible dimer,

wherein the first C2c1 or C2c3 fusion construct is operably linked toone or more nuclear localization signals,

wherein the second C2c1 or C2c3 fusion construct is operably linked toone or more nuclear export signals,

wherein contact with an inducer energy source brings the first andsecond halves of the inducible dimer together,

wherein bringing the first and second halves of the inducible dimertogether allows the first and second C2c1 fusion constructs toconstitute a functional C2c1 CRISPR-Cas system or the first and secondC2c3 fusion constructs to constitute a functional C2c3 CRISPR-Cassystem,

wherein the C2c1 CRISPR-Cas system or C2c3 CRISPR-Cas system comprises aguide RNA (gRNA) comprising a guide sequence capable of hybridizing to atarget sequence in a genomic locus of interest in a cell, and

wherein the functional C2c1 CRISPR-Cas system or the functional C2c3CRISPR-Cas system binds to the target sequence and, optionally, editsthe genomic locus to alter gene expression.

In an aspect of the invention in the inducible C2c1 CRISPR-Cas system,the inducible dimer is or comprises or consists essentially of orconsists of an inducible heterodimer. In an aspect, in inducible C2c1CRISPR-Cas system, the first half or a first portion or a first fragmentof the inducible heterodimer is or comprises or consists of or consistsessentially of an FKBP, optionally FKBP12. In an aspect of theinvention, in the inducible C2c1 CRISPR-Cas system, the second half or asecond portion or a second fragment of the inducible heterodimer is orcomprises or consists of or consists essentially of FRB. In an aspect ofthe invention, in the inducible C2c1 CRISPR-Cas system, the arrangementof the first C2c1 fusion construct is or comprises or consists of orconsists essentially of N′ terminal C2c1 part-FRB-NES. In an aspect ofthe invention, in the inducible C2c1 CRISPR-Cas system, the arrangementof the first C2c1 fusion construct is or comprises or consists of orconsists essentially of NES-N′ terminal C2c1 part-FRB-NES. In an aspectof the invention, in the inducible C2c1 CRISPR-Cas system, thearrangement of the second C2c1 fusion construct is or comprises orconsists essentially of or consists of C′ terminal C2c1 part-FKBP-NLS.In an aspect the invention provides in the inducible C2c1 CRISPR-Cassystem, the arrangement of the second C2c1 fusion construct is orcomprises or consists of or consists essentially of NLS-C′ terminal C2c1part-FKBP-NLS. In an aspect, in inducible C2c1 CRISPR-Cas system therecan be a linker that separates the C2c1 part from the half or portion orfragment of the inducible dimer. In an aspect, in the inducible C2c1CRISPR-Cas system, the inducer energy source is or comprises or consistsessentially of or consists of rapamycin. In an aspect, in inducible C2c1CRISPR-Cas system, the inducible dimer is an inducible homodimer. In anaspect, in inducible C2c1 CRISPR-Cas system, the C2c1 is AacC2c1. In anaspect, in the inducible C2c1 CRISPR-Cas system, one or more functionaldomains are associated with one or both parts of the C2c1, e.g., thefunctional domains optionally including a transcriptional activator, atranscriptional or a nuclease such as a Fok1 nuclease. In an aspect, inthe inducible C2c1 CRISPR-Cas system, the functional C2c1 CRISPR-Cassystem binds to the target sequence and the enzyme is a dead-C2c1,optionally having a diminished nuclease activity of at least 970/, or100% (or no more than 3% and advantageously 0% nuclease activity) ascompared with the C2c1 not having the at least one mutation. Theinvention further comprehends and an aspect of the invention provides, apolynucleotide encoding the inducible C2c1 CRISPR-Cas system as hereindiscussed.

In an aspect of the invention in the inducible C2c3 CRISPR-Cas system,the inducible dimer is or comprises or consists essentially of orconsists of an inducible heterodimer. In an aspect, in inducible C2c3CRISPR-Cas system, the first half or a first portion or a first fragmentof the inducible heterodimer is or comprises or consists of or consistsessentially of an FKBP, optionally FKBP12. In an aspect of theinvention, in the inducible C2c3 CRISPR-Cas system, the second half or asecond portion or a second fragment of the inducible heterodimer is orcomprises or consists of or consists essentially of FRB. In an aspect ofthe invention, in the inducible C2c3 CRISPR-Cas system, the arrangementof the first C2c3 fusion construct is or comprises or consists of orconsists essentially of N′ terminal C2c3 part-FRB-NES. In an aspect ofthe invention, in the inducible C2c3 CRISPR-Cas system, the arrangementof the first C2c3 fusion construct is or comprises or consists of orconsists essentially of NES-N′ terminal C2c3 part-FRB-NES. In an aspectof the invention, in the inducible C2c3 CRISPR-Cas system, thearrangement of the second C2c3 fusion construct is or comprises orconsists essentially of or consists of C′ terminal C2c3 part-FKBP-NLS.In an aspect the invention provides in the inducible C2c3 CRISPR-Cassystem, the arrangement of the second C2c3 fusion construct is orcomprises or consists of or consists essentially of NLS-C′ terminal C2c3part-FKBP-NLS. In an aspect, in inducible C2c3 CRISPR-Cas system therecan be a linker that separates the C2c3 part from the half or portion orfragment of the inducible dimer. In an aspect, in the inducible C2c3CRISPR-Cas system, the inducer energy source is or comprises or consistsessentially of or consists of rapamycin. In an aspect, in inducible C2c3CRISPR-Cas system, the inducible dimer is an inducible homodimer. In anaspect, in the inducible C2c3 CRISPR-Cas system, one or more functionaldomains are associated with one or both parts of the C2c3, e.g., thefunctional domains optionally including a transcriptional activator, atranscriptional or a nuclease such as a Fok1 nuclease. In an aspect, inthe inducible C2c3 CRISPR-Cas system, the functional C2c3 CRISPR-Cassystem binds to the target sequence and the enzyme is a dead-C2c3,optionally having a diminished nuclease activity of at least 97/6, or100% (or no more than 3% and advantageously 0% nuclease activity) ascompared with the C2c3 not having the at least one mutation. Theinvention further comprehends and an aspect of the invention provides, apolynucleotide encoding the inducible C2c3 CRISPR-Cas system as hereindiscussed.

In an aspect, the invention provides a vector for delivery of the firstC2c1 fusion construct, attached to a first half or portion or fragmentof an inducible dimer and operably linked to one or more nuclearlocalization signals, according as herein discussed. In an aspect, theinvention provides a vector for delivery of the second C2c1 fusionconstruct, attached to a second half or portion or fragment of aninducible dimer and operably linked to one or more nuclear exportsignals.

In an aspect, the invention provides a vector for delivery of the firstC2c3 fusion construct, attached to a first half or portion or fragmentof an inducible dimer and operably linked to one or more nuclearlocalization signals, according as herein discussed. In an aspect, theinvention provides a vector for delivery of the second C2c3 fusionconstruct, attached to a second half or portion or fragment of aninducible dimer and operably linked to one or more nuclear exportsignals.

In an aspect, the invention provides a vector for delivery of both: thefirst C2c1 fusion construct, attached to a first half or portion orfragment of an inducible dimer and operably linked to one or morenuclear localization signals, as herein discussed; and the second C2c1fusion construct, attached to a second half or portion or fragment of aninducible dimer and operably linked to one or more nuclear exportsignals, as herein discussed.

In an aspect, the invention provides a vector for delivery of both: thefirst C2c3 fusion construct, attached to a first half or portion orfragment of an inducible dimer and operably linked to one or morenuclear localization signals, as herein discussed; and the second C2c3fusion construct, attached to a second half or portion or fragment of aninducible dimer and operably linked to one or more nuclear exportsignals, as herein discussed.

In an aspect, the vector can be single plasmid or expression cassette.

The invention, in an aspect, provides a eukaryotic host cell or cellline transformed with any of the vectors herein discussed or expressingthe inducible C2c1 or inducible C2c3 CRISPR-Cas system as hereindiscussed.

The invention, in an aspect provides, a transgenic organism transformedwith any of the vectors herein discussed or expressing the inducibleC2c1 or inducible C2c3 CRISPR-Cas system herein discussed, or theprogeny thereof. In an aspect, the invention provides a model organismwhich constitutively expresses the inducible C2c1 or C2c3 CRISPR-Cassystem as herein discussed.

In an aspect, the invention provides non-naturally occurring orengineered inducible C2c1 CRISPR-Cas system, comprising:

a first C2c1 fusion construct attached to a first half of an inducibleheterodimer and

a second C2c1 fusion construct attached to a second half of theinducible heterodimer,

wherein the first C2c1 fusion construct is operably linked to one ormore nuclear localization signals,

wherein the second C2c1 fusion construct is operably linked to a nuclearexport signal,

wherein contact with an inducer energy source brings the first andsecond halves of the inducible heterodimer together,

wherein bringing the first and second halves of the inducibleheterodimer together allows the first and second C2c1 fusion constructsto constitute a functional C2c1 CRISPR-Cas system,

wherein the C2c1 CRISPR-Cas system comprises a guide RNA (gRNA)comprising a guide sequence capable of hybridizing to a target sequencein a genomic locus of interest in a cell, and

wherein the functional C2c1 CRISPR-Cas system edits the genomic locus toalter gene expression.

In an aspect, the invention provides non-naturally occurring orengineered inducible C2c3 CRISPR-Cas system, comprising:

a first C2c3 fusion construct attached to a first half of an inducibleheterodimer and

a second C2c3 fusion construct attached to a second half of theinducible heterodimer,

wherein the first C2c3 fusion construct is operably linked to one ormore nuclear localization signals,

wherein the second C2c3 fusion construct is operably linked to a nuclearexport signal,

wherein contact with an inducer energy source brings the first andsecond halves of the inducible heterodimer together,

wherein bringing the first and second halves of the inducibleheterodimer together allows the first and second C2c3 fusion constructsto constitute a functional C2c3 CRISPR-Cas system,

wherein the C2c3 CRISPR-Cas system comprises a guide RNA (gRNA)comprising a guide sequence capable of hybridizing to a target sequencein a genomic locus of interest in a cell, and

wherein the functional C2c3 CRISPR-Cas system edits the genomic locus toalter gene expression.

In an aspect, the invention provides a method of treating a subject inneed thereof, comprising inducing gene editing by transforming thesubject with the polynucleotide as herein discussed or any of thevectors herein discussed and administering an inducer energy source tothe subject. The invention comprehends uses of such a polynucleotide orvector in the manufacture of a medicament, e.g., such a medicament fortreating a subject or for such a method of treating a subject. Theinvention comprehends the polynucleotide as herein discussed or any ofthe vectors herein discussed for use in a method of treating a subjectin need thereof comprising inducing gene editing, wherein the methodfurther comprises administering an inducer energy source to the subject.In an aspect, in the method, a repair template is also provided, forexample delivered by a vector comprising said repair template.

The invention also provides a method of treating a subject in needthereof, comprising inducing transcriptional activation or repression bytransforming the subject with the polynucleotide herein discussed or anyof the vectors herein discussed, wherein said polynucleotide or vectorencodes or comprises the catalytically inactive C2c1 or inactive C2c3and one or more associated functional domains as herein discussed; themethod further comprising administering an inducer energy source to thesubject. The invention also provides the polynucleotide herein discussedor any of the vectors herein discussed for use in a method of treating asubject in need thereof comprising inducing transcriptional activationor repression, wherein the method further comprises administering aninducer energy source to the subject.

Accordingly, the invention comprehends inter alia homodimers as well asheterodimers, dead-C2c1 or C2c1 having essentially no nuclease activity,e.g., through mutation, systems or complexes wherein there is one ormore NLS and/or one or more NES; functional domain(s) linked to splitC2c1; methods, including methods of treatment, and uses.

Accordingly, the invention comprehends inter alia homodimers as well asheterodimers, dead-C2c3 or C2c3 having essentially no nuclease activity,e.g., through mutation, systems or complexes wherein there is one ormore NLS and/or one or more NES; functional domain(s) linked to splitC2c3; methods, including methods of treatment, and uses.

It will be appreciated that where reference is made herein to C2c1, C2c1protein or C2c1 enzyme, this includes the present split C2c1. In oneaspect, the invention provides a method for altering or modifyingexpression of a gene product. The said method may comprise introducinginto a cell containing and expressing a DNA molecule encoding the geneproduct an engineered, non-naturally occurring C2c1 CRISPR-Cas systemcomprising a C2c1 protein and guide RNA that targets the DNA molecule,whereby the guide RNA targets the DNA molecule encoding the gene productand the C2c1 protein cleaves the DNA molecule encoding the gene product,whereby expression of the gene product is altered; and, wherein the C2c1protein and the guide RNA do not naturally occur together. The inventioncomprehends the guide RNA comprising a guide sequence linked to a directrepeat (DR) sequence. The invention further comprehends the C2c1 proteinbeing codon optimized for expression in a eukaryotic cell. In apreferred embodiment the eukaryotic cell is a mammalian cell and in amore preferred embodiment the mammalian cell is a human cell. In afurther embodiment of the invention, the expression of the gene productis decreased.

It will be appreciated that where reference is made herein to C2c3, C2c3protein or C2c3 enzyme, this includes the present split C2c3. In oneaspect, the invention provides a method for altering or modifyingexpression of a gene product. The said method may comprise introducinginto a cell containing and expressing a DNA molecule encoding the geneproduct an engineered, non-naturally occurring C2c3 CRISPR-Cas systemcomprising a C2c3 protein and guide RNA that targets the DNA molecule,whereby the guide RNA targets the DNA molecule encoding the gene productand the C2c3 protein cleaves the DNA molecule encoding the gene product,whereby expression of the gene product is altered; and, wherein the C2c3protein and the guide RNA do not naturally occur together. The inventioncomprehends the guide RNA comprising a guide sequence linked to a directrepeat (DR) sequence. The invention further comprehends the C2c3 proteinbeing codon optimized for expression in a eukaryotic cell. In apreferred embodiment the eukaryotic cell is a mammalian cell and in amore preferred embodiment the mammalian cell is a human cell. In afurther embodiment of the invention, the expression of the gene productis decreased.

In one aspect, the invention provides an engineered, non-naturallyoccurring C2c1 CRISPR-Cas system comprising a C2c1 protein and a guideRNA that targets a DNA molecule encoding a gene product in a cell,whereby the guide RNA targets the DNA molecule encoding the gene productand the C2c1 protein cleaves the DNA molecule encoding the gene product,whereby expression of the gene product is altered; and, wherein the C2c1protein and the guide RNA do not naturally occur together; thisincluding the present split C2c1. The invention comprehends the guideRNA comprising a guide sequence linked to a DR sequence. The inventionfurther comprehends the C2c1 protein being codon optimized forexpression in a eukaryotic cell. In a preferred embodiment theeukaryotic cell is a mammalian cell and in a more preferred embodimentthe mammalian cell is a human cell. In a further embodiment of theinvention, the expression of the gene product is decreased.

In one aspect, the invention provides an engineered, non-naturallyoccurring C2c3 CRISPR-Cas system comprising a C2c3 protein and a guideRNA that targets a DNA molecule encoding a gene product in a cell,whereby the guide RNA targets the DNA molecule encoding the gene productand the C2c3 protein cleaves the DNA molecule encoding the gene product,whereby expression of the gene product is altered; and, wherein the C2c3protein and the guide RNA do not naturally occur together; thisincluding the present split C2c3. The invention comprehends the guideRNA comprising a guide sequence linked to a DR sequence. The inventionfurther comprehends the C2c3 protein being codon optimized forexpression in a eukaryotic cell. In a preferred embodiment theeukaryotic cell is a mammalian cell and in a more preferred embodimentthe mammalian cell is a human cell. In a further embodiment of theinvention, the expression of the gene product is decreased.

In another aspect, the invention provides an engineered, non-naturallyoccurring vector system comprising one or more vectors comprising afirst regulatory element operably linked to a C2c1 or a C2c3 CRISPR-Cassystem guide RNA that targets a DNA molecule encoding a gene product anda second regulatory element operably linked to a C2c1 protein or a C2c3;this includes the present split C2c1 or C2c3. Components (a) and (b) maybe located on same or different vectors of the system. The guide RNAtargets the DNA molecule encoding the gene product in a cell and theC2c1 protein or C2c3 cleaves the DNA molecule encoding the gene product,whereby expression of the gene product is altered; and, wherein the C2c1protein or the C2c3 and the guide RNA do not naturally occur together.The invention comprehends the guide RNA comprising a guide sequencelinked to a DR sequence. The invention further comprehends the C2c1protein or C2c3 being codon optimized for expression in a eukaryoticcell. In a preferred embodiment the eukaryotic cell is a mammalian celland in a more preferred embodiment the mammalian cell is a human cell.In a further embodiment of the invention, the expression of the geneproduct is decreased.

In one aspect, the invention provides a vector system comprising one ormore vectors. In some embodiments, the system comprises: (a) a firstregulatory element operably linked to a DR sequence and one or moreinsertion sites for inserting one or more guide sequences downstream ofthe DR sequence, wherein when expressed, the guide sequence directssequence-specific binding of a C2c1 or a C2c3 CRISPR-Cas complex to atarget sequence in a eukaryotic cell, wherein the C2c1 or C2c3CRISPR-Cas complex comprises C2c1 or C2c3 complexed with (1) the guidesequence that is hybridized to the target sequence, and (2) the DRsequence; and (b) a second regulatory element operably linked to anenzyme-coding sequence encoding said C2c1 or C2c3 enzyme comprising anuclear localization sequence; wherein components (a) and (b) arelocated on the same or different vectors of the system; this includesthe present split C2c1 or C2c3. In some embodiments, component (a)further comprises two or more guide sequences operably linked to thefirst regulatory element, wherein when expressed, each of the two ormore guide sequences direct sequence specific binding of a C2c1 or aC2c3 CRISPR-Cas complex to a different target sequence in a eukaryoticcell.

In some embodiments, the C2c1 or C2c3 CRISPR-Cas complex comprises oneor more nuclear localization sequences of sufficient strength to driveaccumulation of said C2c1 or C2c3 CRISPR-Cas complex in a detectableamount in the nucleus of a eukaryotic cell. Without wishing to be boundby theory, it is believed that a nuclear localization sequence is notnecessary for C2c1 or C2c3 CRISPR-Cas complex activity in eukaryotes,but that including such sequences enhances activity of the system,especially as to targeting nucleic acid molecules in the nucleus.

In some embodiments, the C2c1 enzyme is C2c1 of a bacterial speciesselected from the group consisting of Alicyclobacillus acidoterrestris(e.g., ATCC 49025), Alicyclobacillus contaminans (e.g., DSM 17975) (Ac,Desulfovibrio inopinatus (e.g., DSM 10711), Desulfonatronumthiodismutans (e.g., strain MLF-1), Opitutaceae bacterium TAV5,Tuberibacillus calidus (e.g., DSM 17572), Bacillus thermoamylovorans(e.g., strain B4166), Brevibacillus sp. CF112, Bacillus sp. NSP2.1,Desulfatirhabdium butyrativorans (e.g., DSM 18734), Alicyclobacillusherbarius (e.g., DSM 13609), Citrobacter freundii (e.g., ATCC 8090),Brevibacillus agri (e.g., BAB-2500), and Methylobacterium nodulans(e.g., ORS 2060). The enzyme may be a C2c1 or C2c3 homolog or ortholog.In some embodiments, the C2c1 or C2c3 is codon-optimized for expressionin a eukaryotic cell. In some embodiments, the C2c1 or C2c3 directscleavage of one or two strands at the location of the target sequence.In a preferred embodiment, the strand break is a staggered cut with a 5′overhang. In some embodiments, the first regulatory element is apolymerase III promoter. In some embodiments, the second regulatoryelement is a polymerase II promoter. In some embodiments, the directrepeat has a minimum length of 16 nts and a single stem loop. In furtherembodiments the direct repeat has a length longer than 16 nts,preferably more than 17 nts, and has more than one stem loop oroptimized secondary structures.

In one aspect, the invention provides a eukaryotic host cell comprising(a) a first regulatory element operably linked to a direct repeatsequence and one or more insertion sites for inserting one or more guidesequences downstream of the DR sequence, wherein when expressed, theguide sequence directs sequence-specific binding of a C2c1 or C2c3CRISPR-Cas complex to a target sequence in a eukaryotic cell, whereinthe C2c1 or C2c3 CRISPR-Cas complex comprises C2c1 or C2c3 complexedwith (1) the guide sequence that is hybridized to the target sequence,and (2) the DR sequence; and/or (b) a second regulatory element operablylinked to an enzyme-coding sequence encoding said C2c1 or C2c3 enzymecomprising a nuclear localization sequence. In some embodiments, thehost cell comprises components (a) and (b); this includes the presentsplit C2c1 or split C2c3. In some embodiments, component (a), component(b), or components (a) and (b) are stably integrated into a genome ofthe host eukaryotic cell. In some embodiments, component (a) furthercomprises two or more guide sequences operably linked to the firstregulatory element, wherein when expressed, each of the two or moreguide sequences direct sequence specific binding of a C2c1 or C2c3CRISPR-Cas complex to a different target sequence in a eukaryotic cell.In some embodiments, the C2c1 or C2c3 is codon-optimized for expressionin a eukaryotic cell. In some embodiments, the C2c1 or C2c3 directscleavage of one or two strands at the location of the target sequence.In a preferred embodiment, the strand break is a staggered cut with a 5′overhang. In some embodiments, the C2c1 or C2c3 lacks DNA strandcleavage activity. In some embodiments, the first regulatory element isa polymerase III promoter. In some embodiments, the direct repeat has aminimum length of 16 nts and a single stem loop. In further embodimentsthe direct repeat has a length longer than 16 nts, preferably more than17 nts, and has more than one stem loop or optimized secondarystructures. In an aspect, the invention provides a non-human eukaryoticorganism; preferably a multicellular eukaryotic organism, comprising aeukaryotic host cell according to any of the described embodiments. Inother aspects, the invention provides a eukaryotic organism; preferablya multicellular eukaryotic organism, comprising a eukaryotic host cellaccording to any of the described embodiments. The organism in someembodiments of these aspects may be an animal; for example a mammal.Also, the organism may be an arthropod such as an insect. The organismalso may be a plant. Further, the organism may be a fungus.

In one aspect, the invention provides a kit comprising one or more ofthe components described herein. In some embodiments, the kit comprisesa vector system and instructions for using the kit. In some embodiments,the vector system comprises (a) a first regulatory element operablylinked to a direct repeat sequence and one or more insertion sites forinserting one or more guide sequences downstream of the DR sequence,wherein when expressed, the guide sequence directs sequence-specificbinding of a C2c1 CRISPR-Cas complex to a target sequence in aeukaryotic cell, wherein the C2c1 or C2c3 CRISPR-Cas complex comprisesC2c1 or C2c3 complexed with (1) the guide sequence that is hybridized tothe target sequence, and (2) the DR sequence; and/or (b) a secondregulatory element operably linked to an enzyme-coding sequence encodingsaid C2c1 or C2c3 enzyme comprising a nuclear localization sequence andadvantageously this includes the present split C2c1 or split C2c3. Insome embodiments, the kit comprises components (a) and (b) located onthe same or different vectors of the system. In some embodiments,component (a) further comprises two or more guide sequences operablylinked to the first regulatory element, wherein when expressed, each ofthe two or more guide sequences direct sequence specific binding of aC2c1 or C2c3 CRISPR-Cas complex to a different target sequence in aeukaryotic cell. In some embodiments, the C2c1 or C2c3 comprises one ormore nuclear localization sequences of sufficient strength to driveaccumulation of said C2c1 or C2c3 in a detectable amount in the nucleusof a eukaryotic cell. In some embodiments, the C2c1 enzyme is C2c1 of abacterial species selected from the group consisting of Alicyclobacillusacidoterrestris (e.g., ATCC 49025), Alicyclobacillus contaminans (e.g.,DSM 17975), Desulfovibrio inopinatus (e.g., DSM 10711), Desulfonatronumthiodismutans (e.g., strain MLF-1), Opitutaceae bacterium TAV5,Tuberibacillus calidus (e.g., DSM 17572), Bacillus thermoamylovorans(e.g., strain B4166), Brevibacillus sp. CF112, Bacillus sp. NSP2.1,Desulfatirhabdium butyrativorans (e.g., DSM 18734), Alicyclobacillusherbarius (e.g., DSM 13609), Citrobacter freundii (e.g., ATCC 8090),Brevibacillus agri (e.g., BAB-2500), Methylobacterium nodulans (e.g.,ORS 2060). The enzyme may be a C2c1 or C2c3 homolog or ortholog. In someembodiments, the C2c1 or C2c3 is codon-optimized for expression in aeukaryotic cell. In some embodiments, the C2c1 or C2c3 directs cleavageof one or two strands at the location of the target sequence. In apreferred embodiment, the strand break is a staggered cut with a 5′overhang. In some embodiments, the CRISPR enzyme lacks DNA strandcleavage activity. In some embodiments, the direct repeat has a minimumlength of 16 nts and a single stem loop. In further embodiments thedirect repeat has a length longer than 16 nts, preferably more than 17nts, and has more than one stem loop or optimized secondary structures.

In one aspect, the invention provides a method of modifying a targetpolynucleotide in a eukaryotic cell. In some embodiments, the methodcomprises allowing a C2c1 or C2c3 CRISPR-Cas complex to bind to thetarget polynucleotide to effect cleavage of said target polynucleotidethereby modifying the target polynucleotide, wherein the C2c1 or C2c3CRISPR-Cas complex comprises C2c1 or C2c3 complexed with a guidesequence hybridized to a target sequence within said targetpolynucleotide, wherein said guide sequence is linked to a direct repeatsequence. In some embodiments, said cleavage comprises cleaving one ortwo strands at the location of the target sequence by said C2c1 or C2c3;this includes the present split C2c1 or split C2c3. In some embodiments,said cleavage results in decreased transcription of a target gene. Insome embodiments, the method further comprises repairing said cleavedtarget polynucleotide by homologous recombination with an exogenoustemplate polynucleotide, wherein said repair results in a mutationcomprising an insertion, deletion, or substitution of one or morenucleotides of said target polynucleotide. In some embodiments, saidmutation results in one or more amino acid changes in a proteinexpressed from a gene comprising the target sequence. In someembodiments, the method further comprises delivering one or more vectorsto said eukaryotic cell, wherein the one or more vectors driveexpression of one or more of: the C2c1 or C2c3, and the guide sequencelinked to the DR sequence. In some embodiments, said vectors aredelivered to the eukaryotic cell in a subject. In some embodiments, saidmodifying takes place in said eukaryotic cell in a cell culture. In someembodiments, the method further comprises isolating said eukaryotic cellfrom a subject prior to said modifying. In some embodiments, the methodfurther comprises returning said eukaryotic cell and/or cells derivedtherefrom to said subject.

In one aspect, the invention provides a method of modifying expressionof a polynucleotide in a eukaryotic cell. In some embodiments, themethod comprises allowing a C2c1 or C2c3 CRISPR-Cas complex to bind tothe polynucleotide such that said binding results in increased ordecreased expression of said polynucleotide; wherein the C2c1 or C2c3CRISPR-Cas complex comprises C2c1 or C2c3 complexed with a guidesequence hybridized to a target sequence within said polynucleotide,wherein said guide sequence is linked to a direct repeat sequence; thisincludes the present split C2c1 or split C2c3. In some embodiments, themethod further comprises delivering one or more vectors to saideukaryotic cells, wherein the one or more vectors drive expression ofone or more of: the C2c1 or C2c3, and the guide sequence linked to theDR sequence.

In one aspect, the invention provides a method of generating a modeleukaryotic cell comprising a mutated disease gene. In some embodiments,a disease gene is any gene associated an increase in the risk of havingor developing a disease. In some embodiments, the method comprises (a)introducing one or more vectors into a eukaryotic cell, wherein the oneor more vectors drive expression of one or more of: C2c1 or C2c3, and aguide sequence linked to a direct repeat sequence; and (b) allowing aC2c1 or C2c3 CRISPR-Cas complex to bind to a target polynucleotide toeffect cleavage of the target polynucleotide within said disease gene,wherein the C2c1 or C2c3 CRISPR-Cas complex comprises the C2c1 or C2c3complexed with (1) the guide sequence that is hybridized to the targetsequence within the target polynucleotide, and (2) the DR sequence,thereby generating a model eukaryotic cell comprising a mutated diseasegene; this includes the present split C2c1 or split C2c3. In someembodiments, said cleavage comprises cleaving one or two strands at thelocation of the target sequence by said C2c1 or C2c3. In a preferredembodiment, the strand break is a staggered cut with a 5′ overhang. Insome embodiments, said cleavage results in decreased transcription of atarget gene. In some embodiments, the method further comprises repairingsaid cleaved target polynucleotide by homologous recombination with anexogenous template polynucleotide, wherein said repair results in amutation comprising an insertion, deletion, or substitution of one ormore nucleotides of said target polynucleotide. In some embodiments,said mutation results in one or more amino acid changes in a proteinexpression from a gene comprising the target sequence.

In one aspect, the invention provides a method for developing abiologically active agent that modulates a cell signaling eventassociated with a disease gene. In some embodiments, a disease gene isany gene associated an increase in the risk of having or developing adisease. In some embodiments, the method comprises (a) contacting a testcompound with a model cell of any one of the described embodiments; and(b) detecting a change in a readout that is indicative of a reduction oran augmentation of a cell signaling event associated with said mutationin said disease gene, thereby developing said biologically active agentthat modulates said cell signaling event associated with said diseasegene.

In one aspect, the invention provides a recombinant polynucleotidecomprising a guide sequence downstream of a direct repeat sequence,wherein the guide sequence when expressed directs sequence-specificbinding of a C2c1 or C2c3 CRISPR-Cas complex to a corresponding targetsequence present in a eukaryotic cell. In some embodiments, the targetsequence is a viral sequence present in a eukaryotic cell. In someembodiments, the target sequence is a proto-oncogene or an oncogene.

In one aspect the invention provides for a method of selecting one ormore cell(s) by introducing one or more mutations in a gene in the oneor more cell (s), the method comprising: introducing one or more vectorsinto the cell (s), wherein the one or more vectors drive expression ofone or more of: C2c1 or C2c3, a guide sequence linked to a direct repeatsequence, and an editing template; wherein the editing templatecomprises the one or more mutations that abolish C2c1 or C2c3 cleavage;allowing homologous recombination of the editing template with thetarget polynucleotide in the cell(s) to be selected; allowing a C2c1 orC2c3 CRISPR-Cas complex to bind to a target polynucleotide to effectcleavage of the target polynucleotide within said gene, wherein the C2c1or C2c3 CRISPR-Cas complex comprises the C2c1 or C2c3 complexed with (1)the guide sequence that is hybridized to the target sequence within thetarget polynucleotide, and (2) the direct repeat sequence, whereinbinding of the C2c1 or C2c3 CRISPR-Cas complex to the targetpolynucleotide induces cell death, thereby allowing one or more cell(s)in which one or more mutations have been introduced to be selected; thisincludes the present split C2c1 or split C2c3. In another preferredembodiment of the invention the cell to be selected may be a eukaryoticcell. Aspects of the invention allow for selection of specific cellswithout requiring a selection marker or a two-step process that mayinclude a counter-selection system.

Herein there is the phrase “this includes the present split C2c1 orsplit C2c3” or similar text; and, this is to indicate that C2c1 inembodiments herein can be a split C2c1 and C2c3 in embodiments hereincan be a split C2c3 as herein discussed.

In an aspect the invention involves a non-naturally occurring orengineered inducible C2c1 CRISPR-Cas system, comprising a first C2c1fusion construct attached to a first half of an inducible heterodimerand a second C2c1 fusion construct attached to a second half of theinducible heterodimer, wherein the first C2c1 fusion construct isoperably linked to one or more nuclear localization signals, wherein thesecond C2c1 fusion construct is operably linked to a nuclear exportsignal, wherein contact with an inducer energy source brings the firstand second halves of the inducible heterodimer together, whereinbringing the first and second halves of the inducible heterodimertogether allows the first and second C2c1 fusion constructs toconstitute a functional C2c1 CRISPR-Cas system, wherein the C2c1CRISPR-Cas system comprises a guide RNA (gRNA) comprising a guidesequence capable of hybridizing to a target sequence in a genomic locusof interest in a cell, and wherein the functional C2c1 CRISPR-Cas systemedits the genomic locus to alter gene expression. In an embodiment ofthe invention the first half of the inducible heterodimer is FKBP12 andthe second half of the inducible heterodimer is FRB. In anotherembodiment of the invention the inducer energy source is rapamycin.

In an aspect the invention involves a non-naturally occurring orengineered inducible C2c3 CRISPR-Cas system, comprising a first C2c3fusion construct attached to a first half of an inducible heterodimerand a second C2c3 fusion construct attached to a second half of theinducible heterodimer, wherein the first C2c3 fusion construct isoperably linked to one or more nuclear localization signals, wherein thesecond C2c3 fusion construct is operably linked to a nuclear exportsignal, wherein contact with an inducer energy source brings the firstand second halves of the inducible heterodimer together, whereinbringing the first and second halves of the inducible heterodimertogether allows the first and second C2c3 fusion constructs toconstitute a functional C2c3 CRISPR-Cas system, wherein the C2c3CRISPR-Cas system comprises a guide RNA (gRNA) comprising a guidesequence capable of hybridizing to a target sequence in a genomic locusof interest in a cell, and wherein the functional C2c3 CRISPR-Cas systemedits the genomic locus to alter gene expression. In an embodiment ofthe invention the first half of the inducible heterodimer is FKBP12 andthe second half of the inducible heterodimer is FRB. In anotherembodiment of the invention the inducer energy source is rapamycin.

An inducer energy source may be considered to be simply an inducer or adimerizing agent. The term ‘inducer energy source’ is used hereinthroughout for consistency. The inducer energy source (or inducer) actsto reconstitute the C2c1 or C2c3. In some embodiments, the inducerenergy source brings the two parts of the C2c1 or C2c3 together throughthe action of the two halves of the inducible dimer. The two halves ofthe inducible dimer therefore are brought tougher in the presence of theinducer energy source. The two halves of the dimer will not form intothe dimer (dimerize) without the inducer energy source.

Thus, the two halves of the inducible dimer cooperate with the inducerenergy source to dimerize the dimer. This in turn reconstitutes the C2c1or C2c3 by bringing the first and second parts of the C2c1 or C2c3together.

The CRISPR enzyme fusion constructs each comprise one part of the splitC2c1 or split C2c3. These are fused, preferably via a linker such as aGlySer linker described herein, to one of the two halves of the dimer.The two halves of the dimer may be substantially the same two monomersthat together that form the homodimer, or they may be different monomersthat together form the heterodimer. As such, the two monomers can bethought of as one half of the full dimer.

The C2c1 or C2c3 is split in the sense that the two parts of the C2c1 orC2c3 enzyme substantially comprise a functioning C2c1 or C2c3. That C2c1or C2c3 may function as a genome editing enzyme (when forming a complexwith the target DNA and the guide), such as a nickase or a nuclease(cleaving both strands of the DNA), or it may be a dead-C2c1 ordead-C2c3 which is essentially a DNA-binding protein with very little orno catalytic activity, due to typically mutation(s) in its catalyticdomains.

The two parts of the split C2c1 or C2c3 can be thought of as the N′terminal part and the C′ terminal part of the split C2c1 or split C2c3.The fusion is typically at the split point of the C2c1 or C2c3. In otherwords, the C′ terminal of the N′ terminal part of the split C2c1 or C2c3is fused to one of the dimer halves, whilst the N′ terminal of the C′terminal part is fused to the other dimer half.

The C2c1 or C2c3 does not have to be split in the sense that the breakis newly created. The split point is typically designed in silico andcloned into the constructs. Together, the two parts of the split C2c1 orsplit C2c3, the N′ terminal and C′ terminal parts, form a full C2c1 orC2c3, comprising preferably at least 70% or more of the wildtype aminoacids (or nucleotides encoding them), preferably at least 80% or more,preferably at least 90% or more, preferably at least 95% or more, andmost preferably at least 99% or more of the wildtype amino acids (ornucleotides encoding them). Some trimming may be possible, and mutantsare envisaged. Non-functional domains may be removed entirely. What isimportant is that the two parts may be brought together and that thedesired C2c1 or C2c3 function is restored or reconstituted.

The dimer may be a homodimer or a heterodimer.

One or more, preferably two, NLSs may be used in operable linkage to thefirst C2c1 construct. One or more, preferably two, NESs may be used inoperable linkage to the first C2c1 construct. The NLSs and/or the NESspreferably flank the split C2c1-dimer (i.e., half dimer) fusion, i.e.,one NLS may be positioned at the N′ terminal of the first C2c1 constructand one NLS may be at the C′ terminal of the first C2c1 construct.Similarly, one NES may be positioned at the N′ terminal of the secondC2c1 construct and one NES may be at the C′ terminal of the second C2c1construct. Where reference is made to N′ or C′ terminals, it will beappreciated that these correspond to 5′ ad 3′ ends in the correspondingnucleotide sequence.

One or more, preferably two, NLSs may be used in operable linkage to thefirst C2c3 construct. One or more, preferably two, NESs may be used inoperable linkage to the first C2c3 construct. The NLSs and/or the NESspreferably flank the split C2c3-dimer (i.e., half dimer) fusion, i.e.,one NLS may be positioned at the N′ terminal of the first C2c3 constructand one NLS may be at the C′ terminal of the first C2c3 construct.Similarly, one NES may be positioned at the N′ terminal of the secondC2c3 construct and one NES may be at the C′ terminal of the second C2c3construct. Where reference is made to N′ or C′ terminals, it will beappreciated that these correspond to 5′ ad 3′ ends in the correspondingnucleotide sequence.

A preferred arrangement is that the first C2c1 construct is arranged5′-NLS-(N′ terminal C2c1 part)-linker-(first half of the dimer)-NLS-3′or that the first C2c3 construct is arranged 5′-NLS-(N′ terminal C2c3part)-linker-(first half of the dimer)-NLS-3′. A preferred arrangementis that the second C2c1 construct is arranged 5′-NES-(second half of thedimer)-linker-(C′ terminal C2c1 part)-NES-3′ or the second C2c3construct is arranged 5′-NES-(second half of the dimer)-linker-(C′terminal C2c3 part)-NES-3′. A suitable promoter is preferably upstreamof each of these constructs. The two constructs may be deliveredseparately or together.

In some embodiments, one or all of the NES(s) in operable linkage to thesecond C2c1 construct may be swapped out for an NLS. However, this maybe typically not preferred and, in other embodiments, the localizationsignal in operable linkage to the second C2c1 construct is one or moreNES(s). In some embodiments, one or all of the NES(s) in operablelinkage to the second C2c3 construct may be swapped out for an NLS.However, this may be typically not preferred and, in other embodiments,the localization signal in operable linkage to the second C2c3 constructis one or more NES(s)

It will also be appreciated that the NES may be operably linked to theN′ terminal fragment of the split C2c1 or split C2c3 and that the NLSmay be operably linked to the C′ terminal fragment of the split C2c1 orsplit C2c3. However, the arrangement where the NLS is operably linked tothe N′ terminal fragment of the split C2c1 or split C2c3 and that theNES is operably linked to the C′ terminal fragment of the split C2c1 orsplit C2c3 may be preferred.

The NES functions to localize the second C2c1 or C2c3 fusion constructoutside of the nucleus, at least until the inducer energy source isprovided (e.g., at least until an energy source is provided to theinducer to perform its function). The presence of the inducer stimulatesdimerization of the two C2c1 or C2c3 fusions within the cytoplasm andmakes it thermodynamically worthwhile for the dimerized, first andsecond, C2c1 or C2c3 fusions to localize to the nucleus. Without beingbound by theory, Applicants believe that the NES sequesters the secondC2c1 or C2c3 fusion to the cytoplasm (i.e., outside of the nucleus). TheNLS on the first C2c1 or C2c3 fusion localizes it to the nucleus. Inboth cases, Applicants use the NES or NLS to shift an equilibrium (theequilibrium of nuclear transport) to a desired direction. Thedimerization typically occurs outside of the nucleus (a very smallfraction might happen in the nucleus) and the NLSs on the dimerizedcomplex shift the equilibrium of nuclear transport to nuclearlocalization, so the dimerized and hence reconstituted C2c1 or C2c3enters the nucleus.

Beneficially, Applicants are able to reconstitute function in the splitC2c1 or split C2c3. Transient transfection is used to prove the conceptand dimerization occurs in the background in the presence of the inducerenergy source. No activity is seen with separate fragments of the C2c1or C2c3. Stable expression through lentiviral delivery is then used todevelop this and show that a split C2c1 or C2c3 approach can be used.

This present split C2c1 or split C2c3 approach is beneficial as itallows the C2c1 or C2c3 activity to be inducible, thus allowing fortemporal control. Furthermore, different localization sequences may beused (i.e., the NES and NLS as preferred) to reduce background activityfrom auto-assembled complexes. Tissue specific promoters, for exampleone for each of the first and second C2c1 or C2c3 fusion constructs, mayalso be used for tissue-specific targeting, thus providing spatialcontrol. Two different tissue specific promoters may be used to exert afiner degree of control if required. The same approach may be used inrespect of stage-specific promoters or there may a mixture of stage andtissue specific promoters, where one of the first and second C2c1 orC2c3 fusion constructs is under the control of (i.e. operably linked toor comprises) a tissue-specific promoter, whilst the other of the firstand second C2c1 or C2c3 fusion constructs is under the control of (i.e.operably linked to or comprises) a stage-specific promoter.

The inducible C2c1 or C2c3 CRISPR-Cas system comprises one or morenuclear localization sequences (NLSs), as described herein, for exampleas operably linked to the first C2c1 or C2c3 fusion construct. Thesenuclear localization sequences are ideally of sufficient strength todrive accumulation of said first C2c1 or C2c3 fusion construct in adetectable amount in the nucleus of a eukaryotic cell. Without wishingto be bound by theory, it is believed that a nuclear localizationsequence is not necessary for C2c1 or C2c3 CRISPR-Cas complex activityin eukaryotes, but that including such sequences enhances activity ofthe system, especially as to targeting nucleic acid molecules in thenucleus, and assists with the operation of the present 2-part system.

Equally, the second C2c1 or C2c3 fusion construct is operably linked toa nuclear export sequence (NES). Indeed, it may be linked to one or morenuclear export sequences. In other words, the number of export sequencesused with the second C2c1 or C2c3 fusion construct is preferably 1 or 2or 3. Typically 2 is preferred, but 1 is enough and so is preferred insome embodiments. Suitable examples of NLS and NES are known in the art.For example, a preferred nuclear export signal (NES) is human proteintyrosine kinase 2. Preferred signals will be species specific.

Where the FRB and FKBP system are used, the FKBP is preferably flankedby nuclear localization sequences (NLSs). Where the FRB and FKBP systemare used, the preferred arrangement is N′ terminal C2c1-FRB-NES: C′terminal C2c1-FKBP-NLS or N′ terminal C2c3-FRB-NES: C′ terminalC2c3-FKBP-NLS. Thus, the first C2c1 or C2c3 fusion construct wouldcomprise the C′ terminal C2c1 or C2c3 part and the second C2c1 or C2c3fusion construct would comprise the N′ terminal C2c1 or C2c3 part.

Another beneficial aspect to the present invention is that it may beturned on quickly, i.e. that is has a rapid response. It is believed,without being bound by theory, that C2c1 or C2c3 activity can be inducedthrough dimerization of existing (already present) fusion constructs(through contact with the inducer energy source) more rapidly thanthrough the expression (especially translation) of new fusionconstructs. As such, the first and second C2c1 or C2c3 fusion constructsmay be expressed in the target cell ahead of time, i.e. before C2c1 orC2c3 activity is required. C2c1 or C2c3 activity can then be temporallycontrolled and then quickly constituted through addition of the inducerenergy source, which ideally acts more quickly (to dimerize theheterodimer and thereby provide C2c1 or C2c3 activity) than throughexpression (including induction of transcription) of C2c1 or C2c3delivered by a vector, for example.

The terms C2c1 or C2c1 enzyme and CRISPR enzyme are used interchangeablyherein unless otherwise apparent. The terms C2c3 or C2c3 enzyme andCRISPR enzyme are used interchangeably herein unless otherwise apparent.

Applicants demonstrate that C2c1 or C2c3 can be split into twocomponents, which reconstitute a functional nuclease when brought backtogether. Employing rapamycin sensitive dimerization domains, Applicantsgenerate a chemically inducible C2c1 or C2c3 for temporal control ofC2c1-mediated genome editing or C2c3-mediated genome editing andtranscription modulation. Put another way, Applicants demonstrate thatC2c1 or C2c3 can be rendered chemically inducible by being split intotwo fragments and that rapamycin-sensitive dimerization domains may beused for controlled reassembly of the C2c1 or C2c3. Applicants show thatthe re-assembled C2c1 or C2c3 may be used to mediate genome editing(through nuclease/nickase activity) as well as transcription modulation(as a DNA-binding domain, the so-called “dead C2c1” or “dead C2c3”).

As such, the use of rapamycin-sensitive dimerization domains ispreferred. Reassembly of the C2c1 or C2c3 is preferred. Reassembly canbe determined by restoration of binding activity. Where the C2c1 or C2c3is a nickase or induces a double-strand break, suitable comparisonpercentages compared to a wildtype are described herein.

Rapamycin treatments can last 12 days. The dose can be 200 nM. Thistemporal and/or molar dosage is an example of an appropriate dose forHuman embryonic kidney 293FT (HEK293FT) cell lines and this may also beused in other cell lines. This figure can be extrapolated out fortherapeutic use in vivo into, for example, mg/kg. However, it is alsoenvisaged that the standard dosage for administering rapamycin to asubject is used here as well. By the “standard dosage”, it is meant thedosage under rapamycin's normal therapeutic use or primary indication(i.e. the dose used when rapamycin is administered for use to preventorgan rejection).

It is noteworthy that the preferred arrangement of C2c1-FRB/FKBP orC2c3-FRB/FKBP pieces are separate and inactive until rapamycin-induceddimerization of FRB and FKBP results in reassembly of a functionalfull-length C2c1 or C2c3 nuclease. Thus, it is preferred that first C2c1or C2c3 fusion construct attached to a first half of an inducibleheterodimer is delivered separately and/or is localized separately fromthe second C2c1 or C2c3 fusion construct attached to a first half of aninducible heterodimer.

To sequester the C2c1(N)-FRB fragment or C2c3(N)-FRB fragment in thecytoplasm, where it is less likely to dimerize with thenuclear-localized C2c1(C)-FKBP fragment or C2c3(C)-FKBP fragment, it ispreferable to use on C2c1(N)-FRB a single nuclear export sequence (NES)from the human protein tyrosine kinase 2 (C2c1(N)—FRB-NES) or onC2c3(N)-FRB a single nuclear export sequence (NES) from the humanprotein tyrosine kinase 2 (C2c3(N)—FRB-NES). In the presence ofrapamycin, C2c1(N)—FRB-NES dimerizes with C2c1(C)-FKBP-2×NLS orC2c3(N)—FRB-NES dimerizes with C2c3(C)-FKBP-2×NLS to reconstitute acomplete C2c1 protein or C2c3 protein, which shifts the balance ofnuclear trafficking toward nuclear import and allows DNA targeting.

High dosage of C2c1 or C2c3 can exacerbate indel frequencies atoff-target (OT) sequences which exhibit few mismatches to the guidestrand. Such sequences are especially susceptible, if mismatches arenon-consecutive and/or outside of the seed region of the guide.Accordingly, temporal control of C2c1 or C2c3 activity could be used toreduce dosage in long-term expression experiments and therefore resultin reduced off-target indels compared to constitutively active C2c1 orC2c3.

Viral delivery is preferred. In particular, a lentiviral or AAV deliveryvector is envisaged. Applicants generate a split-C2c1 or split-C2c3lentivirus construct, similar to the lentiCRISPR plasmid. The splitpieces should be small enough to fit the −4.7 kb size limitation of AAV.

Applicants demonstrate that stable, low copy expression of split C2c1 orsplit C2c3 can be used to induce substantial indels at a targeted locuswithout significant mutation at off-target sites. Applicants clone C2c1fragments (2 parts based on split 5, described herein) or C2c3fragments.

A dead C2c1 or C2c3 may also be used, comprising a VP64 transactivationdomain, for example added to C2c1(C)-FKBP-2×NLS(dead-C2c1(C)-FKBP-2×NLS-VP64) or C2c3(C)-FKBP-2×NLS(dead-C2c3(C)-FKBP-2×NLS-VP64). These fragments reconstitute acatalytically inactive C2c1-VP64 fusion (dead-C2c1-VP64) or C2c3-VP64fusion (dead-C2c3-VP64). Transcriptional activation is induced by VP64in the presence of rapamycin to induce the dimerization of theC2c1(C)-FKBP fusion and the C2c1(N)-FRB fusion or the C2c3(C)-FKBPfusion and the C2c3(N)-FRB fusion. In other words, Applicants test theinducibility of split dead-C2c1-VP64 or split dead-C2c3-VP64 and showthat transcriptional activation is induced by split dead-C2c1-VP64 orsplit dead-C2c3-VP64 in the presence of rapamycin. As such, the presentinducible C2c1 or C2c3 may be associated with one or more functionaldomain, such as a transcriptional activator or repressor or a nuclease(such as Fok1). A functional domain may be bound to or fused with onepart of the split C2c1 or split C2c3.

A preferred arrangement is that the first C2c1 construct is arranged5′-First Localization Signal-(N′ terminal C2c1 part)-linker-(first halfof the dimer)-First Localization Signal-3′ or the first C2c3 constructis arranged 5′-First Localization Signal-(N′ terminal C2c3part)-linker-(first half of the dimer)-First Localization Signal-3′, andthe second C2c1 construct is arranged 5′-Second LocalizationSignal-(second half of the dimer)-linker-(C′ terminal C2c1 part)-SecondLocalization Signal-Functional Domain-3′ or the second C2c3 construct isarranged 5′-Second Localization Signal-(second half of thedimer)-linker-(C′ terminal C2c3 part)-Second LocalizationSignal-Functional Domain-3′. Here, a functional domain is placed at the3′ end of the second C2c1 or C2c3 construct. Alternatively, a functionaldomain may be placed at the 5′ end of the first C2c1 or C2c3 construct.One or more functional domains may be used at the 3′ end or the 5′ endor at both ends. A suitable promoter is preferably upstream of each ofthese constructs. The two constructs may be delivered separately ortogether. The Localization Signals may be an NLS or an NES, so long asthey are not inter-mixed on each construct.

In an aspect the invention provides an inducible C2c1 or C2c3 CRISPR-Cassystem wherein the C2c1 or C2c3 has a diminished nuclease activity of atleast 97/a, or 100% as compared with the C2c1 or C2c3 enzyme not havingthe at least one mutation.

Accordingly, it is also preferred that the C2c1 or C2c3 is a dead-C2c1or dead-C2c3. Ideally, the split should always be so that the catalyticdomain(s) are unaffected. For the dead-C2c1 or dead-C2c3 the intentionis that DNA binding occurs, but not cleavage or nickase activity isshown.

In an aspect the invention provides an inducible C2c1 or C2c3 CRISPR-Cassystem as herein discussed wherein one or more functional domains isassociated with the C2c1 or C2c3. This functional domain may beassociated with (i.e. bound to or fused with) one part of the split C2c1or both or one part of the split C2c3 or both. There may be oneassociated with each of the two parts of the split C2c1 or C2c3. Thesemay therefore be typically provided as part of the first and/or secondC2c1 or C2c3 fusion constructs, as fusions within that construct. Thefunctional domains are typically fused via a linker, such as GlySerlinker, as discussed herein. The one or more functional domains may betranscriptional activation domain or a repressor domain. Although theymay be different domains it is preferred that all the functional domainsare either activator or repressor and that a mixture of the two is notused.

The transcriptional activation domain may comprise VP64, p65, MyoD1,HSF1, RTA or SET7/9.

In an aspect, the invention provides an inducible C2c1 or C2c3CRISPR-Cas system as herein discussed wherein the one or more functionaldomains associated with the C2c1 or C2c3 is a transcriptional repressordomain.

In an aspect, the invention provides an inducible C2c1 or C2c3CRISPR-Cas system as herein discussed wherein the transcriptionalrepressor domain is a KRAB domain.

In an aspect, the invention provides an inducible C2c1 or C2c3CRISPR-Cas system as herein discussed wherein the transcriptionalrepressor domain is a NuE domain, NcoR domain, SID domain or a SID4Xdomain.

In an aspect the invention provides an inducible C2c1 or C2c3 CRISPR-Cassystem as herein discussed wherein the one or more functional domainsassociated with the adaptor protein have one or more activitiescomprising methylase activity, demethylase activity, transcriptionactivation activity, transcription repression activity, transcriptionrelease factor activity, histone modification activity, RNA cleavageactivity, DNA cleavage activity, DNA integration activity or nucleicacid binding activity.

Histone modifying domains are also preferred in some embodiments.Exemplary histone modifying domains are discussed below. Transposasedomains, HR (Homologous Recombination) machinery domains, recombinasedomains, and/or integrase domains are also preferred as the presentfunctional domains. In some embodiments, DNA integration activityincludes HR machinery domains, integrase domains, recombinase domainsand/or transposase domains.

In an aspect the invention provides an inducible C2c1 or C2c3 CRISPR-Cassystem as herein discussed wherein the DNA cleavage activity is due to anuclease.

In an aspect the invention provides an inducible C2c1 or C2c3 CRISPR-Cassystem as herein discussed wherein the nuclease comprises a Fok1nuclease.

The use of such functional domains, which are preferred with the presentsplit C2c1 or split C2c3 system, is also discussed in detail inKonermann et al. (“Genome-scale transcriptional activation with anengineered CRISPR-Cas9 complex” Nature published 11 Dec. 2014).

The present system may be used with any guide.

Modified guides may be used in certain embodiments. Particularlypreferred are guides embodying the teachings of Konermann Nature 11 Dec.2014 paper mentioned above. These guides are modified so thatprotein-binding RNA portions (such as aptamers) are added. Suchportion(s) may replace a portion of the guide. Corresponding RNA-bindingprotein domains can be used to then recognise the RNA and recruitfunctional domains, such as those described herein, to the guide. Thisis primarily for use with dead-C2c1 or dead-C2c3 leading totranscriptional activation or repression or DNA cleavage throughnucleases such as Fok1. The use of such guides in combination withdead-C2c1 or C2c3 is powerful, and it is especially powerful if the C2c1or C2c3 itself is also associated with its own functional domain, asdiscussed herein. When a dead-C2c1 or dead-C2c3 (with or without its ownassociated functional domain) is induced to reconstitute in accordancewith the present invention, i.e. is a split C2c1 or split C2c3, then thetool is especially useful.

A guide RNA (gRNA), also preferred for use in the present invention, cancomprise a guide sequence capable of hybridizing to a target sequence ina genomic locus of interest in a cell, wherein the gRNA is modified bythe insertion of distinct RNA sequence(s) that bind to one or moreadaptor proteins, and wherein the adaptor protein is associated with oneor more functional domains. The C2c1 or C2c3 may comprise at least onemutation, such that the C2c1 or C2c3 enzyme has no more than 5% of thenuclease activity of the C2c1 or C2c3 enzyme not having the at least onemutation; and/or at least one or more nuclear localization sequences.Also provided is a non-naturally occurring or engineered compositioncomprising: one or more guide RNA (gRNA) comprising a guide sequencecapable of hybridizing to a target sequence in a genomic locus ofinterest in a cell, a C2c1 or C2c3 enzyme comprising at least one ormore nuclear localization sequences, wherein the C2c1 or C2c3 enzymecomprises at least one mutation, such that the C2c1 enzyme or C2c3enzyme has no more than 5% of the nuclease activity of the C2c1 enzymeor C2c3 enzyme not having the at least one mutation, wherein the atleast one gRNA is modified by the insertion of distinct RNA sequence(s)that bind to one or more adaptor proteins, and wherein the adaptorprotein is associated with one or more functional domains.

The gRNA that is preferably modified by the insertion of distinct RNAsequence(s) that bind to one or more adaptor proteins. The insertion ofdistinct RNA sequence(s) that bind to one or more adaptor proteins ispreferably an aptamer sequence or two or more aptamer sequences specificto the same or different adaptor protein(s). The adaptor proteinpreferably comprises MS2, PP7, Qp, F2, GA, fr, JP501, M12, R17, BZ13,JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205,4Cb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s, PRR1. Cell lines stably expressinginter alia split dead-C2c1 or split dead-C2c3 can be useful.

Applicants demonstrate that C2c1 or C2c3 can be split into two distinctfragments, which reconstitute a functional full-length C2c1 or C2c3nuclease when brought back together using chemical induction. The splitC2c1 or split C2c3 architecture will be useful for a variety ofapplications. For example, split C2c1 may enable genetic strategies forrestricting C2c1 activity to intersectional cell populations by puttingeach fragment under a different tissue specific promoter or split C2c3may enable genetic strategies for restricting C2c3 activity tointersectional cell populations by putting each fragment under adifferent tissue specific promoter. Additionally, different chemicallyinducible dimerization domains such as APA and gibberellin may also beemployed.

The inducer energy source is preferably chemical induction.

The split position or location is the point at which the first part ofthe C2c1 or C2c3 enzyme is separated from the second part. In someembodiments, the first part will comprise or encode amino acids 1 to X,whilst the second part will comprise or encode amino acids X+1 to theend. In this example, the numbering is contiguous, but this may notalways be necessary as amino acids (or the nucleotides encoding them)could be trimmed from the end of either of the split ends, provided thatsufficient DNA binding activity and, if required, DNA nickase orcleavage activity is retained, for example at least 40%, 50%, 60%, 70%,80%, 90% or 95% activity compared to wildtype C2c1 or wildtype C2c3.

The exemplary numbering provided herein may be in reference to thewildtype protein, preferably the wildtype C2c1 or wildtype C2c3.However, it is envisaged that mutants of the wildtype C2c1, such as ofBacillus C2c1 protein, or mutants of the wildtype C2c3 can be used. Thenumbering may also not follow exactly the C2c1 or C2c3 numbering as, forinstance, some N′ or C′ terminal truncations or deletions may be used,but this can be addressed using standard sequence alignment tools.Orthologs are also preferred as a sequence alignment tool.

Thus, the split position may be selected using ordinary skill in theart, for instance based on crystal data and/or computational structurepredictions.

Ideally, the split position should be located within a region or loop.Preferably, the split position occurs where an interruption of the aminoacid sequence does not result in the partial or full destruction of astructural feature (e.g. alpha-helixes or beta-sheets). Unstructuredregions (regions that do not show up in the crystal structure becausethese regions are not structured enough to be “frozen” in a crystal) areoften preferred options. Applicants can for example make splits inunstructured regions that are exposed on the surface of C2c1 or C2c3.

Applicants can follow the following procedure which is provided as apreferred example and as guidance. Since unstructured regions don't showup in the crystal structure, Applicants cross-reference the surroundingamino acid sequence of the crystal with the primary amino acid sequenceof the C2c1 or C2c3. Each unstructured region can be made of for exampleabout 3 to 10 amino acids, which does not show up in the crystal.Applicants therefore make the split in between these amino acids. Toinclude more potential split sides Applicants include splits located inloops at the outside of C2c1 or C2c3 using the same criteria as withunstructured regions.

In some embodiments, the split position is in an outside loop of theC2c1 or C2c3. In other preferred embodiments, the split position is inan unstructured region of the C2c1 or C2c3. An unstructured region istypically a highly flexible outside loop whose structure cannot bereadily determined from a crystal pattern.

Once the split position has been identified, suitable constructs can bedesigned.

Typically, an NES is positioned at the N′ terminal end of the first partof the split amino acid (or the 5′ end of nucleotide encoding it). Inthat case, an NLS is positioned at the C′ terminal end of the secondpart of the split amino acid (or the 3′ end of the nucleotide encodingit). In this way, the first C2c1 or C2c3 fusion construct may beoperably linked to one or more nuclear export signals and the secondC2c1 or C2c3 fusion construct may be operably linked to a nuclearlocalization signal.

Of course, the reverse arrangement may be provided, where an NLS ispositioned at the N′ terminal end of the first part of the split aminoacid (or the 5′ end of nucleotide encoding it). In that case, an NES ispositioned at the C′ terminal end of the second part of the split aminoacid (or the 3′ end of the nucleotide encoding it). Thus, the first C2c1or C2c3 fusion construct may be operably linked to one or more nuclearlocalization signals and the second C2c1 or C2c3 fusion construct may beoperably linked to a nuclear export signal.

Splits which keep the two parts (either side of the split) roughly thesame length may be advantageous for packing purposes. For example, it isthought to be easier to maintain stoichiometry between both pieces whenthe transcripts are about the same size.

In certain examples, the N- and C-term pieces of human codon-optimizedC2c1 such as C2c1 are fused to FRB and FKBP dimerization domains,respectively. This arrangement may be preferred. They may be switchedover (i.e. N′ term to FKBP and C′ term to FRB). In certain examples, theN- and C-term pieces of human codon-optimized C2c3 such as C2c3 arefused to FRB and FKBP dimerization domains, respectively. Thisarrangement may be preferred. They may be switched over (i.e. N′ term toFKBP and C′ term to FRB).

Linkers such as (GGGGS)₃ (SEQ ID NO: 18) are preferably used herein toseparate the C2c1 fragment or C2c3 fragment from the dimerizationdomain. (GGGGS)₃ (SEQ ID NO: 18) is preferable because it is arelatively long linker (15 amino acids). The glycine residues are themost flexible and the serine residues enhance the chance that the linkeris on the outside of the protein. (GGGGS)₆ (SEQ ID NO: 19) (GGGGS)₉ (SEQID NO: 20) or (GGGGS)₁₂ (SEQ ID NO: 21) may preferably be used asalternatives. Other preferred alternatives are (GGGGS)₁ (SEQ ID NO: 22),(GGGGS)₂ (SEQ ID NO: 23), (GGGGS)₄ (SEQ ID NO: 24), (GGGGS)₅ (SEQ ID NO:25), (GGGGS), (SEQ ID NO: 26), (GGGGS)₅ (SEQ ID NO: 27), (GGGGS)₁₀ (SEQID NO: 28), or (GGGGS)₁₁ (SEQ ID NO: 29).

For example, (GGGGS)₃ (SEQ ID NO: 18) may be included between the N′term C2c1 fragment or C2c3 fragment and FRB. For example, (GGGGS)₃ (SEQID NO: 18) may be included between FKB and the C′ term C2c1 fragment orC2c3 fragment.

Alternative linkers are available, but highly flexible linkers arethought to work best to allow for maximum opportunity for the 2 parts ofthe C2c1 or C2c3 to come together and thus reconstitute C2c1 or C2c3activity. One alternative is that the NLS of nucleoplasmin can be usedas a linker.

A linker can also be used between the C2c1 or C2c3 and any functionaldomain. Again, a (GGGGS)₃ (SEQ ID NO: 18) linker may be used here (orthe 6 (SEQ ID NO: 19), 9 (SEQ ID NO: 20), or 12 (SEQ ID NO: 21) repeatversions therefore) or the NLS of nucleoplasmin can be used as a linkerbetween C2c1 or C2c3 and the functional domain.

Alternatives to the FRB/FKBP system are envisaged. For example the ABAand gibberellin system.

Accordingly, preferred examples of the FKBP family are any one of thefollowing inducible systems. FKBP which dimerizes with CalcineurinA(CNA), in the presence of FK506; FKBP which dimerizes with CyP-Fas, inthe presence of FKCsA; FKBP which dimerizes with FRB, in the presence ofRapamycin; GyrB which dimerizes with GryB, in the presence ofCoumermycin; GAI which dimerizes with GID1, in the presence ofGibberellin; or Snap-tag which dimerizes with HaloTag, in the presenceof HaXS.

Alternatives within the FKBP family itself are also preferred. Forexample, FKBP, which homo-dimerizes (i.e. one FKBP dimerizes withanother FKBP) in the presence of FK1012. Thus, also provided is anon-naturally occurring or engineered inducible C2c1 or C2c3 CRISPR-Cassystem, comprising:

a first C2c1 fusion construct or a first C2c3 fusion construct attachedto a first half of an inducible homodimer and

a second C2c1 fusion construct or a second C2c3 fusion constructattached to a second half of the inducible homodimer,

wherein the first C2c1 fusion construct or the first C2c3 fusionconstruct is operably linked to one or more nuclear localizationsignals,

wherein the second C2c1 fusion construct or the second C2c3 fusionconstruct is operably linked to a (optionally one or more) nuclearexport signal(s),

wherein contact with an inducer energy source brings the first andsecond halves of the inducible homodimer together,

wherein bringing the first and second halves of the inducible homodimertogether allows the first and second C2c1 fusion constructs toconstitute a functional C2c1 CRISPR-Cas system or the first and secondC2c3 fusion constructs to constitute a functional C2c3 CRISPR-Cassystem,

wherein the C2c1 or C2c3 CRISPR-Cas system comprises a guide RNA (gRNA)comprising a guide sequence capable of hybridizing to a target sequencein a genomic locus of interest in a cell, and

wherein the functional C2c1 or C2c3 CRISPR-Cas system binds to thetarget sequence and, optionally, edits the genomic locus to alter geneexpression.

In one embodiment, the homodimer is preferably FKBP and the inducerenergy source is preferably FK1012. In another embodiment, the homodimeris preferably GryB and the inducer energy source is preferablyCoumermycin. In another embodiment, the homodimer is preferably ABA andthe inducer energy source is preferably Gibberellin.

In other embodiments, the dimer is a heterodimer. Preferred examples ofheterodimers are any one of the following inducible systems: FKBP whichdimerizes with CalcineurinA (CNA), in the presence of FK506; FKBP whichdimerizes with CyP-Fas, in the presence of FKCsA; FKBP which dimerizeswith FRB, in the presence of Rapamycin, in the presence of Coumermycin;GAI which dimerizes with GID1, in the presence of Gibberellin; orSnap-tag which dimerizes with HaloTag, in the presence of HaXS.

Applicants used FKBP/FRB because it is well characterized and bothdomains are sufficiently small (<100 amino acids) to assist withpackaging. Furthermore, rapamycin has been used for a long time and sideeffects are well understood. Large dimerization domains (>300 aa) shouldwork too but may require longer linkers to make enable C2c1 or C2c3reconstitution.

Paulmurugan and Gambhir (Cancer Res, Aug. 15, 2005 65; 7413) discussesthe background to the FRB/FKBP/Rapamycin system. Another useful paper isthe article by Crabtree et al. (Chemistry & Biology 13, 99-107, January2006).

In an example, a single vector, an expression cassette (plasmid) isconstructed. gRNA is under the control of a U6 promoter. Two differentC2c1 or C2c3 splits are used. The split C2c1 or C2c3 construct is basedon a first C2c1 or C2c3 fusion construct, flanked by NLSs, with FKBPfused to C terminal part of the split C2c1 or split C2c3 via a GlySerlinker; and a second C2c1 or C2c3 fusion construct, flanked by NESs,with FRB fused with the N terminal part of the split C2c1 or split C2c3via a GlySer linker. To separate the first and second C2c1 or C2c3fusion constructs, P2A is used splitting on transcription. The SplitC2c1 or split C2c3 shows indel formation similar to wildtype in thepresence of rapamycin, but markedly lower indel formation than thewildtype in the absence of rapamycin.

Accordingly, a single vector is provided. The vector comprises:

a first C2c1 fusion construct or a first C2c3 fusion construct attachedto a first half of an inducible dimer and

a second C2c1 fusion construct or a second C2c3 fusion constructattached to a second half of the inducible dimer,

wherein the first C2c1 fusion construct or the first C2c3 fusionconstruct is operably linked to one or more nuclear localizationsignals,

wherein the second C2c1 fusion construct or the second C2c3 fusionconstruct is operably linked to one or more nuclear export signals,

wherein contact with an inducer energy source brings the first andsecond halves of the inducible heterodimer together,

wherein bringing the first and second halves of the inducibleheterodimer together allows the first and second C2c1 fusion constructsto constitute a functional C2c1 CRISPR-Cas system or the first andsecond C2c3 fusion constructs to constitute a functional C2c3 CRISPR-Cassystem,

wherein the C2c1 or C2c3 CRISPR-Cas system comprises a guide RNA (gRNA)comprising a guide sequence capable of hybridizing to a target sequencein a genomic locus of interest in a cell, and

wherein the functional C2c1 or C2c3 CRISPR-Cas system binds to thetarget sequence and, optionally, edits the genomic locus to alter geneexpression. These elements are preferably provided on a singleconstruct, for example an expression cassette.

The first C2c1 fusion construct or the first C2c3 fusion construct ispreferably flanked by at least one nuclear localization signal at eachend. The second C2c1 fusion construct or the second C2c3 fusionconstruct is preferably flanked by at least one nuclear export signal ateach end.

Also provided is a method of treating a subject in need thereof,comprising inducing gene editing by transforming the subject with thepolynucleotide encoding the system or any of the present vectors andadministering an inducer energy source to the subject. A suitable repairtemplate may also be provided, for example delivered by a vectorcomprising said repair template.

Also provided is a method of treating a subject in need thereof,comprising inducing transcriptional activation or repression bytransforming the subject with the polynucleotide encoding the presentsystem or any of the present vectors, wherein said polynucleotide orvector encodes or comprises the catalytically inactive C2c1 or C2c3 andone or more associated functional domains; the method further comprisingadministering an inducer energy source to the subject.

Compositions comprising the present system for use in said method oftreatment are also provided. Use of the present system in themanufacture of a medicament for such methods of treatment are alsoprovided.

Examples of conditions treatable by the present system are describedherein or in documents cited herein.

The single vector can comprise a transcript-splitting agent, for exampleP2A. P2A splits the transcript in two, to separate the first and secondC2c1 fusion constructs or the first and second C2c3 fusion constructs.The splitting is due to “ribosomal skipping”. In essence, the ribosomeskips an amino acid during translation, which breaks the protein chainand results in two separate polypeptides/proteins. The single vector isalso useful for applications where low background activity is not ofconcern but a high inducible activity is desired.

One example would be the generation of clonal embryonic stem cell lines.The normal procedure is transient transfection with plasmids encodingwildtype C2c1 or C2c1 nickases or wildtype C2c3 or C2c3 nickases. Theseplasmids produce C2c1 or C2c3 molecules, which stay active for severaldays and have a higher chance of off target activity. Using the singleexpression vector for split C2c1 or C2c3 allows restricting “high” C2c1or C2c3 activity to a shorter time window (e.g. one dose of an inducer,such as rapamycin). Without continual (daily) inducer (e.g. rapamycin)treatments the activity of single expression split C2c1 or C2c3 vectorsis low and presents a reduced chance of causing unwanted off targeteffects.

A peak of induced C2c1 or C2c3 activity is beneficial in someembodiments and may most easily be brought about using a single deliveryvector, but it is also possible through a dual vector system (eachvector delivering one half of the split C2c1 or C2c3). The peak may behigh activity and for a short timescale, typically the lifetime of theinducer.

Accordingly, provided is a method for generation of clonal embryonicstem cell lines, comprising transfecting one or more embryonic stemcells with a polynucleotide encoding the present system or one of thepresent vectors to express the present split C2c1 or C2c3 andadministering or contacting the one or more stem cells with the presentinducer energy source to induce reconstitution of the C2c1 or C2c3. Arepair template may be provided.

As with all methods described herein, it will be appreciated thatsuitable gRNA or guides will be required.

Where functional domains and the like are “associated” with one or otherpart of the enzyme, these are typically fusions. The term “associatedwith” is used here in respect of how one molecule ‘associates’ withrespect to another, for example between parts of the C2c1 and afunctional domain or the C2c3 and a functional domain. In the case ofsuch protein-protein interactions, this association may be viewed interms of recognition in the way an antibody recognises an epitope.Alternatively, one protein may be associated with another protein via afusion of the two, for instance one subunit being fused to anothersubunit. Fusion typically occurs by addition of the amino acid sequenceof one to that of the other, for instance via splicing together of thenucleotide sequences that encode each protein or subunit. Alternatively,this may essentially be viewed as binding between two molecules ordirect linkage, such as a fusion protein. In any event, the fusionprotein may include a linker between the two subunits of interest (i.e.between the enzyme and the functional domain or between the adaptorprotein and the functional domain). Thus, in some embodiments, the partof the C2c1 or C2c3 is associated with a functional domain by bindingthereto. In other embodiments, the C2c1 or C2c3 is associated with afunctional domain because the two are fused together, optionally via anintermediate linker. Examples of linkers include the GlySer linkersdiscussed herein.

Other examples of inducers include light and hormones. For light, theinducible dimers may be heterodimers and include first light-induciblehalf of a dimer and a second (and complimentary) light-inducible half ofa dimer. A preferred example of first and second light-inducible dimerhalves is the CIB1 and CRY2 system. The CIB1 domain is a heterodimericbinding partner of the light-sensitive Cryptochrome 2 (CRY2).

In another example, the blue light-responsive Magnet dimerization system(pMag and nMag) may be fused to the two parts of a split C2c1 or splitC2c3 protein. In response to light stimulation, pMag and nMag dimerizeand C2c1 or C2c3 reassembles. For example, such system is described inconnection with Cas9 in Nihongaki et al. (Nat. Biotechnol. 33, 755-790,2015).

The invention comprehends that the inducer energy source may be heat,ultrasound, electromagnetic energy or chemical. In a preferredembodiment of the invention, the inducer energy source may be anantibiotic, a small molecule, a hormone, a hormone derivative, a steroidor a steroid derivative. In a more preferred embodiment, the inducerenergy source maybe abscisic acid (ABA), doxycycline (DOX), cumate,rapamycin, 4-hydroxytamoxifen (4OHT), estrogen or ecdysone. Theinvention provides that the at least one switch may be selected from thegroup consisting of antibiotic based inducible systems, electromagneticenergy based inducible systems, small molecule based inducible systems,nuclear receptor based inducible systems and hormone based induciblesystems. In a more preferred embodiment the at least one switch may beselected from the group consisting of tetracycline (Tet)/DOX induciblesystems, light inducible systems, ABA inducible systems, cumaterepressor/operator systems, 4OHT/estrogen inducible systems,ecdysone-based inducible systems and FKBP12/FRAP (FKBP12-rapamycincomplex) inducible systems. Such inducers are also discussed herein andin PCT/US2013/051418, incorporated herein by reference.

In general, any use that can be made of a C2c1 or C2c3, whether wt,nickase or a dead-C2c1/C2c3 (with or without associated functionaldomains) can be pursued using the present split C2c1 or split C2c3approach. The benefit remains the inducible nature of the C2c1 or C2c3activity.

As a further example, split C2c1 or C2c3 fusions with fluorescentproteins like GFP can be made. This would allow imaging of genomic loci(see “Dynamic Imaging of Genomic Loci in Living Human Cells by anOptimized CRISPR/Cas System” Chen B et al. Cell 2013), but in aninducible manner. As such, in some embodiments, one or more of the C2c1or C2c3 parts may be associated (and in particular fused with) afluorescent protein, for example GFP.

Further experiments address whether there is a difference in off-targetcutting, between wild type (wt) and split C2c1 or wild type and splitC2c3, when on-target cutting is at the same level. To do this,Applicants use transient transfection of wt and split C2c1 plasmids orwt and split C2c3 plasmids and harvest at different time points.Applicants look for off-target activation after finding a set of sampleswhere on-target cutting is within +/−5%. Applicants make cell lines withstable expression of wt or split enzyme (C2c1 or C2c3) without guides(using lentivirus). After antibiotic selection, guides are deliveredwith a separate lentivirus and there is harvest at different time pointsto measure on-/off-target cutting.

Applicants introduce a destabilizing sequence (PEST, see “Use of mRNA-and protein-destabilizing elements to develop a highly responsivereporter system” Voon D C et al. Nucleic Acids Research 2005) into theFRB(N)C2c1-NES fragment or the FRB(N)C2c3-NES fragment to facilitatefaster degradation and therefore reduced stability of the splitdead-C2c1-VP64 complex or the split dead-C2c3-VP64 complex.

Such destabilizing sequences as described elsewhere in thisspecification (including PEST) can be advantageous for use with splitC2c1 or split C2c3 systems.

Cell lines stably expressing split dead-C2c1-VP64 and MS2-p65-HSF1+guide are generated. A PLX resistance screen can demonstrate that anon-reversible, timed transcriptional activation can be useful in drugscreens. This approach is may be advantageous when a splitdead-C2c1-VP64 is not reversible.

Cell lines stably expressing split dead-C2c3-VP64 and MS2-p65-HSF1+guide are generated. A PLX resistance screen can demonstrate that anon-reversible, timed transcriptional activation can be useful in drugscreens. This approach is may be advantageous when a splitdead-C2c3-VP64 is not reversible.

In one aspect the invention provides a non-naturally occurring orengineered C2c1 or C2c3 CRISPR-Cas system which may comprise at leastone switch wherein the activity of said C2c1 or C2c3 CRISPR-Cas systemis controlled by contact with at least one inducer energy source as tothe switch. In an embodiment of the invention the control as to the atleast one switch or the activity of said C2c1 or C2c3 CRISPR-Cas systemmay be activated, enhanced, terminated or repressed. The contact withthe at least one inducer energy source may result in a first effect anda second effect. The first effect may be one or more of nuclear import,nuclear export, recruitment of a secondary component (such as aneffector molecule), conformational change (of protein, DNA or RNA),cleavage, release of cargo (such as a caged molecule or a co-factor),association or dissociation. The second effect may be one or more ofactivation, enhancement, termination or repression of the control as tothe at least one switch or the activity of said C2c1 or C2c3 CRISPR-Cassystem. In one embodiment the first effect and the second effect mayoccur in a cascade.

In another aspect of the invention the C2c1 or C2c3 CRISPR-Cas systemmay further comprise at least one or more nuclear localization signal(NLS), nuclear export signal (NES), functional domain, flexible linker,mutation, deletion, alteration or truncation. The one or more of theNLS, the NES or the functional domain may be conditionally activated orinactivated. In another embodiment, the mutation may be one or more of amutation in a transcription factor homology region, a mutation in a DNAbinding domain (such as mutating basic residues of a basic helix loophelix), a mutation in an endogenous NLS or a mutation in an endogenousNES. The invention comprehends that the inducer energy source may beheat, ultrasound, electromagnetic energy or chemical. In a preferredembodiment of the invention, the inducer energy source may be anantibiotic, a small molecule, a hormone, a hormone derivative, a steroidor a steroid derivative. In a more preferred embodiment, the inducerenergy source maybe abscisic acid (ABA), doxycycline (DOX), cumate,rapamycin, 4-hydroxytamoxifen (4OHT), estrogen or ecdysone. Theinvention provides that the at least one switch may be selected from thegroup consisting of antibiotic based inducible systems, electromagneticenergy based inducible systems, small molecule based inducible systems,nuclear receptor based inducible systems and hormone based induciblesystems. In a more preferred embodiment the at least one switch may beselected from the group consisting of tetracycline (Tet)/DOX induciblesystems, light inducible systems, ABA inducible systems, cumaterepressor/operator systems, 4OHT/estrogen inducible systems,ecdysone-based inducible systems and FKBP12/FRAP (FKBP12-rapamycincomplex) inducible systems.

Aspects of control as detailed in this application relate to at leastone or more switch(es). The term “switch” as used herein refers to asystem or a set of components that act in a coordinated manner to affecta change, encompassing all aspects of biological function such asactivation, repression, enhancement or termination of that function. Inone aspect the term switch encompasses genetic switches which comprisethe basic components of gene regulatory proteins and the specific DNAsequences that these proteins recognize. In one aspect, switches relateto inducible and repressible systems used in gene regulation. Ingeneral, an inducible system may be off unless there is the presence ofsome molecule (called an inducer) that allows for gene expression. Themolecule is said to “induce expression”. The manner by which thishappens is dependent on the control mechanisms as well as differences incell type. A repressible system is on except in the presence of somemolecule (called a corepressor) that suppresses gene expression. Themolecule is said to “repress expression”. The manner by which thishappens is dependent on the control mechanisms as well as differences incell type. The term “inducible” as used herein may encompass all aspectsof a switch irrespective of the molecular mechanism involved.Accordingly a switch as comprehended by the invention may include but isnot limited to antibiotic based inducible systems, electromagneticenergy based inducible systems, small molecule based inducible systems,nuclear receptor based inducible systems and hormone based induciblesystems. In preferred embodiments the switch may be a tetracycline(Tet)/DOX inducible system, a light inducible systems, a Abscisic acid(ABA) inducible system, a cumate repressor/operator system, a4OHT/estrogen inducible system, an ecdysone-based inducible systems or aFKBP12/FRAP (FKBP12-rapamycin complex) inducible system.

The present C2c1 or C2c3 CRISPR-Cas system may be designed to modulateor alter expression of individual endogenous genes in a temporally andspatially precise manner. The C2c1 or C2c3 CRISPR-Cas system may bedesigned to bind to the promoter sequence of the gene of interest tochange gene expression. The C2c1 or C2c3 may be spilt into two where onehalf is fused to one half of the cryptochrome heterodimer(cryptochrome-2 or CIB1), while the remaining cryptochrome partner isfused to the other half of the C2c1 or C2c3. In some aspects, atranscriptional effector domain may also be included in the C2c1 or C2c3CRISPR-Cas system. Effector domains may be either activators, such asVP16, VP64, or p65, or repressors, such as KRAB, EnR, or SID. Inunstimulated state, the one half C2c1-cryptochrome2 protein orC2c3-cryptochrome2 protein localizes to the promoter of the gene ofinterest, but is not bound to the CIB1-effector protein. Uponstimulation with blue spectrum light, cryptochrome-2 becomes activated,undergoes a conformational change, and reveals its binding domain. CIB1,in turn, binds to cryptochrome-2 resulting in localization of the secondhalf of the C2c1 or the second half of the C2c3 to the promoter regionof the gene of interest and initiating genome editing which may resultin gene overexpression or silencing. Aspects of LITEs are furtherdescribed in Liu, H et al., Science, 2008 and Kennedy M et al., NatureMethods 2010, the contents of which are herein incorporated by referencein their entirety.

Activator and repressor domains which may further modulate function maybe selected on the basis of species, strength, mechanism, duration,size, or any number of other parameters. Preferred effector domainsinclude, but are not limited to, a transposase domain, integrase domain,recombinase domain, resolvase domain, invertase domain, protease domain,DNA methyltransferase domain, DNA demethylase domain, histone acetylasedomain, histone deacetylases domain, nuclease domain, repressor domain,activator domain, nuclear-localization signal domains,transcription-protein recruiting domain, cellular uptake activityassociated domain, nucleic acid binding domain or antibody presentationdomain.

There are several different ways to generate chemical inducible systemsas well: 1. ABI-PYL based system inducible by Abscisic Acid (ABA) (see,e.g., website atstke.sciencemag.org/cgi/content/abstract/sigtrans;4/164/rs2), 2.FKBP-FRB based system inducible by rapamycin (or related chemicals basedon rapamycin) (see, e.g., website atnature.com/nmeth/joumal/v2/n6/full/nmeth763.html), 3. GID1-GAI basedsystem inducible by Gibberellin (GA) (see, e.g., website atnature.com/nchembio/joumal/v8/n5/full/nchembio.922.html).

Another system contemplated by the present invention is a chemicalinducible system based on change in sub-cellular localization.Applicants also comprehend an inducible C2c1 or C2c3 CRISPR-Cas systemengineered to target a genomic locus of interest wherein the C2c1 orC2c3 enzyme is split into two fusion constructs that are further linkedto different parts of a chemical or energy sensitive protein. Thischemical or energy sensitive protein will lead to a change in thesub-cellular localization of either half of the C2c1 or C2c3 enzyme(i.e. transportation of either half of the C2c1 or C2c3 enzyme fromcytoplasm into the nucleus of the cells) upon the binding of a chemicalor energy transfer to the chemical or energy sensitive protein. Thistransportation of fusion constructs from one sub-cellular compartmentsor organelles, in which its activity is sequestered due to lack ofsubstrate for the reconstituted C2c1 or C2c3 CRISPR-Cas system, intoanother one in which the substrate is present would allow the componentsto come together and reconstitute functional activity and to then comein contact with its desired substrate (i.e. genomic DNA in the mammaliannucleus) and result in activation or repression of target geneexpression.

Other inducible systems are contemplated such as, but not limited to,regulation by heavy-metals [Mayo K E et al., Cell 1982, 29:99-108;Searle P F et al., Mol Cell Biol. 1985, 5:1480-1489 and Brinster R L etal., Nature (London) 1982, 296:39-421, steroid hormones [Hynes N E etal., Proc Natl Acad Sci USA 1981, 78:2038-2042; Klock G et al., Nature(London) 1987, 329:734-736 and Lee F et al., Nature (London) 1981,294:228-232.1, heat shock [Nouer L: Heat Shock Response. Boca Raton,Fla.: CRC; 19911 and other reagents have been developed [Mullick A,Massie B: Transcription, translation and the control of gene expression.In Encyclopedia of Cell Technology Edited by: Speir R E. Wiley;2000:1140-1164 and Fussenegger M, Biotechnol Prog 2001, 17:1-51].However, there are limitations with these inducible mammalian promoterssuch as “leakiness” of the “off” state and pleiotropic effects ofinducers (heat shock, heavy metals, glucocorticoids etc.). The use ofinsect hormones (ecdysone) has been proposed in an attempt to reduce theinterference with cellular processes in mammalian cells [No D et al.,Proc Natl Acad Sci USA 1996, 93:3346-3351]. Another elegant system usesrapamycin as the inducer [Rivera V M et al., Nat Med 1996, 2:1028-10321but the role of rapamycin as an immunosuppressant was a major limitationto its use in vivo and therefore it was necessary to find a biologicallyinert compound [Saez E et al., Proc Natl Acad Sci USA 2000,97:14512-14517] for the control of gene expression.

In particular embodiments, the gene editing systems described herein areplaced under the control of a passcode kill switch, which is amechanisms which efficiently kills the host cell when the conditions ofthe cell are altered. This is ensured by introducing hybrid LacI-GalRfamily transcription factors, which require the presence of IPTG to beswitched on (Chan et al. 2015 Nature Nature Chemical Biologydoi:10.1038/nchembio.1979 which can be used to drive a gene encoding anenzyme critical for cell-survival. By combining different transcriptionfactors sensitive to different chemicals, a “code” can be generated.This system can be used to spatially and temporally control the extentof CRISPR-induced genetic modifications, which can be of interest indifferent fields including therapeutic applications and may also be ofinterest to avoid the “escape” of GMOs from their intended environment.

Delivery Generally

Gene Editing or Altering a Target Loci with C2c1 or C2c3

The double strand break or single strand break in one of the strandsadvantageously should be sufficiently close to target position such thatcorrection occurs. In an embodiment, the distance is not more than 50,100, 200, 300, 350 or 400 nucleotides. While not wishing to be bound bytheory, it is believed that the break should be sufficiently close totarget position such that the break is within the region that is subjectto exonuclease-mediated removal during end resection. If the distancebetween the target position and a break is too great, the mutation maynot be included in the end resection and, therefore, may not becorrected, as the template nucleic acid sequence may only be used tocorrect sequence within the end resection region.

In an embodiment, in which a guide RNA and a Type V/Type VI molecule, inparticular C2c1/C2c3 or an ortholog or homolog thereof, preferably aC2c1 or C2c3 nuclease induce a double strand break for the purpose ofinducing HDR-mediated correction, the cleavage site is between 0-200 bp(e.g., 0 to 175, 0 to 150, 0 to 125, 0 to 100, 0 to 75, 0 to 50, 0 to25, 25 to 200, 25 to 175, 25 to 150, 25 to 125, 25 to 100, 25 to 75, 25to 50, 50 to 200, 50 to 175, 50 to 150, 50 to 125, 50 to 100, 50 to 75,75 to 200, 75 to 175, 75 to 150, 75 to 1 25, 75 to 100 bp) away from thetarget position. In an embodiment, the cleavage site is between 0-100 bp(e.g., 0 to 75, 0 to 50, 0 to 25, 25 to 100, 25 to 75, 25 to 50, 50 to100, 50 to 75 or 75 to 100 bp) away from the target position. In afurther embodiment, two or more guide RNAs complexing with C2c1 or C2c3or an ortholog or homolog thereof, may be used to induce multiplexedbreaks for purpose of inducing HDR-mediated correction.

The homology arm should extend at least as far as the region in whichend resection may occur, e.g., in order to allow the resected singlestranded overhang to find a complementary region within the donortemplate. The overall length could be limited by parameters such asplasmid size or viral packaging limits. In an embodiment, a homology armmay not extend into repeated elements. Exemplary homology arm lengthsinclude a least 50, 100, 250, 500, 750 or 1000 nucleotides.

Target position, as used herein, refers to a site on a target nucleicacid or target gene (e.g., the chromosome) that is modified by a TypeV/Type VI, in particular C2c1/C2c3 or an ortholog or homolog thereof,preferably C2c1 or C2c3 molecule-dependent process. For example, thetarget position can be a modified C2c1 or C2c3 molecule cleavage of thetarget nucleic acid and template nucleic acid directed modification,e.g., correction, of the target position. In an embodiment, a targetposition can be a site between two nucleotides, e.g., adjacentnucleotides, on the target nucleic acid into which one or morenucleotides is added. The target position may comprise one or morenucleotides that are altered, e.g., corrected, by a template nucleicacid. In an embodiment, the target position is within a target sequence(e.g., the sequence to which the guide RNA binds). In an embodiment, atarget position is upstream or downstream of a target sequence (e.g.,the sequence to which the guide RNA binds).

A template nucleic acid, as that term is used herein, refers to anucleic acid sequence which can be used in conjunction with a TypeV/Type VI molecule, in particular C2c1/C2c3 or an ortholog or homologthereof, preferably a C2c1 or C2c3 molecule and a guide RNA molecule toalter the structure of a target position. In an embodiment, the targetnucleic acid is modified to have some or all of the sequence of thetemplate nucleic acid, typically at or near cleavage site(s). In anembodiment, the template nucleic acid is single stranded. In analternate embodiment, the template nucleic acid is double stranded. Inan embodiment, the template nucleic acid is DNA, e.g., double strandedDNA. In an alternate embodiment, the template nucleic acid is singlestranded DNA.

In an embodiment, the template nucleic acid alters the structure of thetarget position by participating in homologous recombination. In anembodiment, the template nucleic acid alters the sequence of the targetposition. In an embodiment, the template nucleic acid results in theincorporation of a modified, or non-naturally occurring base into thetarget nucleic acid.

The template sequence may undergo a breakage mediated or catalyzedrecombination with the target sequence. In an embodiment, the templatenucleic acid may include sequence that corresponds to a site on thetarget sequence that is cleaved by an C2c1 or C2c3 mediated cleavageevent. In an embodiment, the template nucleic acid may include sequencethat corresponds to both, a first site on the target sequence that iscleaved in a first C2c1 or C2c3 mediated event, and a second site on thetarget sequence that is cleaved in a second C2c1 or C2c3 mediated event.

In certain embodiments, the template nucleic acid can include sequencewhich results in an alteration in the coding sequence of a translatedsequence, e.g., one which results in the substitution of one amino acidfor another in a protein product, e.g., transforming a mutant alleleinto a wild type allele, transforming a wild type allele into a mutantallele, and/or introducing a stop codon, insertion of an amino acidresidue, deletion of an amino acid residue, or a nonsense mutation. Incertain embodiments, the template nucleic acid can include sequencewhich results in an alteration in a non-coding sequence, e.g., analteration in an exon or in a 5′ or 3′ non-translated or non-transcribedregion. Such alterations include an alteration in a control element,e.g., a promoter, enhancer, and an alteration in a cis-acting ortrans-acting control element.

A template nucleic acid having homology with a target position in atarget gene may be used to alter the structure of a target sequence. Thetemplate sequence may be used to alter an unwanted structure, e.g., anunwanted or mutant nucleotide. The template nucleic acid may includesequence which, when integrated, results in: decreasing the activity ofa positive control element; increasing the activity of a positivecontrol element; decreasing the activity of a negative control element;increasing the activity of a negative control element; decreasing theexpression of a gene; increasing the expression of a gene; increasingresistance to a disorder or disease; increasing resistance to viralentry; correcting a mutation or altering an unwanted amino acid residueconferring, increasing, abolishing or decreasing a biological propertyof a gene product, e.g., increasing the enzymatic activity of an enzyme,or increasing the ability of a gene product to interact with anothermolecule.

The template nucleic acid may include sequence which results in: achange in sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12 or morenucleotides of the target sequence. In an embodiment, the templatenucleic acid may be 20+/−10, 30+/−10, 40+/−10, 50+/−10, 60+/−10,70+/−10, 80+/−10, 90+/−10, 100+/−10, 110+/−10, 120+/−10, 130+/−10,140+/−10, 150+/−10, 160+/−10, 170+/−10, 180+/−10, 190+/−10, 200+/−10,210+/−10, of 220+/−10 nucleotides in length. In an embodiment, thetemplate nucleic acid may be 30+/−20, 40+/−20, 50+/−20, 60+/−20,70+/−20, 80+/−20, 90+/−20, 100+/−20, 110+/−20, 120+/−20, 130+/−20,140+/−20, 150+/−20, 160+/−20, 170+/−20, 180+/−20, 190+/−20, 200+/−20,210+/−20, of 220+/−20 nucleotides in length. In an embodiment, thetemplate nucleic acid is 10 to 1,000, 20 to 900, 30 to 800, 40 to 700,50 to 600, 50 to 500, 50 to 400, 50 to 300, 50 to 200, or 50 to 100nucleotides in length.

A template nucleic acid comprises the following components: [5′ homologyarm]-[replacement sequence]-[3′ homology arm]. The homology arms providefor recombination into the chromosome, thus replacing the undesiredelement, e.g., a mutation or signature, with the replacement sequence.In an embodiment, the homology arms flank the most distal cleavagesites. In an embodiment, the 3′ end of the 5′ homology arm is theposition next to the 5′ end of the replacement sequence. In anembodiment, the 5′ homology arm can extend at least 10, 20, 30, 40, 50,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000nucleotides 5′ from the 5′ end of the replacement sequence. In anembodiment, the 5′ end of the 3′ homology arm is the position next tothe 3′ end of the replacement sequence. In an embodiment, the 3′homology arm can extend at least 10, 20, 30, 40, 50, 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides 3′ from the 3′end of the replacement sequence.

In certain embodiments, one or both homology arms may be shortened toavoid including certain sequence repeat elements. For example, a 5′homology arm may be shortened to avoid a sequence repeat element. Inother embodiments, a 3′ homology arm may be shortened to avoid asequence repeat element. In some embodiments, both the 5′ and the 3′homology arms may be shortened to avoid including certain sequencerepeat elements.

In certain embodiments, a template nucleic acids for correcting amutation may designed for use as a single-stranded oligonucleotide. Whenusing a single-stranded oligonucleotide, 5′ and 3′ homology arms mayrange up to about 200 base pairs (bp) in length, e.g., at least 25, 50,75, 100, 125, 150, 175, or 200 bp in length.

C2c1 or C2c3 Effector Protein Complex System Promoted Non-HomologousEnd-Joining

In certain embodiments, nuclease-induced non-homologous end-joining(NHEJ) can be used to target gene-specific knockouts. Nuclease-inducedNHEJ can also be used to remove (e.g., delete) sequence in a gene ofinterest. Generally, NHEJ repairs a double-strand break in the DNA byjoining together the two ends; however, generally, the original sequenceis restored only if two compatible ends, exactly as they were formed bythe double-strand break, are perfectly ligated. The DNA ends of thedouble-strand break are frequently the subject of enzymatic processing,resulting in the addition or removal of nucleotides, at one or bothstrands, prior to rejoining of the ends. This results in the presence ofinsertion and/or deletion (indel) mutations in the DNA sequence at thesite of the NHEJ repair. Two-thirds of these mutations typically alterthe reading frame and, therefore, produce a non-functional protein.Additionally, mutations that maintain the reading frame, but whichinsert or delete a significant amount of sequence, can destroyfunctionality of the protein. This is locus dependent as mutations incritical functional domains are likely less tolerable than mutations innon-critical regions of the protein. The indel mutations generated byNHEJ are unpredictable in nature; however, at a given break site certainindel sequences are favored and are over represented in the population,likely due to small regions of microhomology. The lengths of deletionscan vary widely; most commonly in the 1-50 bp range, but they can easilybe greater than 50 bp, e.g., they can easily reach greater than about100-200 bp. Insertions tend to be shorter and often include shortduplications of the sequence immediately surrounding the break site.However, it is possible to obtain large insertions, and in these cases,the inserted sequence has often been traced to other regions of thegenome or to plasmid DNA present in the cells.

Because NHEJ is a mutagenic process, it may also be used to delete smallsequence motifs as long as the generation of a specific final sequenceis not required. If a double-strand break is targeted near to a shorttarget sequence, the deletion mutations caused by the NHEJ repair oftenspan, and therefore remove, the unwanted nucleotides. For the deletionof larger DNA segments, introducing two double-strand breaks, one oneach side of the sequence, can result in NHEJ between the ends withremoval of the entire intervening sequence. Both of these approaches canbe used to delete specific DNA sequences; however, the error-pronenature of NHEJ may still produce indel mutations at the site of repair.

Both double strand cleaving Type V/Type VI molecule, in particularC2c1/C2c3 or an ortholog or homolog thereof, preferably C2c1 or C2c3molecules and single strand, or nickase, Type V/Type VI molecule, inparticular C2c1/C2c3 or an ortholog or homolog thereof, preferably C2c1or C2c3 molecules can be used in the methods and compositions describedherein to generate NHEJ-mediated indels. NHEJ-mediated indels targetedto the gene, e.g., a coding region, e.g., an early coding region of agene of interest can be used to knockout (i.e., eliminate expression of)a gene of interest. For example, early coding region of a gene ofinterest includes sequence immediately following a transcription startsite, within a first exon of the coding sequence, or within 500 bp ofthe transcription start site (e.g., less than 500, 450, 400, 350, 300,250, 200, 150, 100 or 50 bp).

In an embodiment, in which a guide RNA and Type V/Type VI molecule, inparticular C2c1/C2c3 or an ortholog or homolog thereof, preferably C2c1or C2c3 nuclease generate a double strand break for the purpose ofinducing NHEJ-mediated indels, a guide RNA may be configured to positionone double-strand break in close proximity to a nucleotide of the targetposition. In an embodiment, the cleavage site may be between 0-500 bpaway from the target position (e.g., less than 500, 400, 300, 200, 100,50, 40, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 bp from thetarget position).

In an embodiment, in which two guide RNAs complexing with Type V/Type VImolecules, in particular C2c1/C2c3 or an ortholog or homolog thereof,preferably C2c1 or C2c3 nickases induce two single strand breaks for thepurpose of inducing NHEJ-mediated indels, two guide RNAs may beconfigured to position two single-strand breaks to provide for NHEJrepair a nucleotide of the target position.

C2c1 or C2c3 Effector Protein Complexes can Deliver Functional Effectors

Unlike CRISPR-Cas-mediated gene knockout, which permanently eliminatesexpression by mutating the gene at the DNA level, CRISPR-Cas knockdownallows for temporary reduction of gene expression through the use ofartificial transcription factors. Mutating key residues in both DNAcleavage domains of the C2c1 or C2c3 protein results in the generationof a catalytically inactive C2c1 or C2c3. A catalytically inactive C2c1or C2c3 complexes with a guide RNA and localizes to the DNA sequencespecified by that guide RNA's targeting domain, however, it does notcleave the target DNA. Fusion of the inactive C2c1 or C2c3 protein to aneffector domain, e.g., a transcription repression domain, enablesrecruitment of the effector to any DNA site specified by the guide RNA.In certain embodiments, C2c1 or C2c3 may be fused to a transcriptionalrepression domain and recruited to the promoter region of a gene.Especially for gene repression, it is contemplated herein that blockingthe binding site of an endogenous transcription factor would aid indownregulating gene expression. In another embodiment, an inactive C2c1or C2c3 can be fused to a chromatin modifying protein. Alteringchromatin status can result in decreased expression of the target gene.

In an embodiment, a guide RNA molecule can be targeted to a knowntranscription response elements (e.g., promoters, enhancers, etc.), aknown upstream activating sequences, and/or sequences of unknown orknown function that are suspected of being able to control expression ofthe target DNA.

In some methods, a target polynucleotide can be inactivated to effectthe modification of the expression in a cell. For example, upon thebinding of a CRISPR complex to a target sequence in a cell, the targetpolynucleotide is inactivated such that the sequence is not transcribed,the coded protein is not produced, or the sequence does not function asthe wild-type sequence does. For example, a protein or microRNA codingsequence may be inactivated such that the protein is not produced.

In certain embodiments, the CRISPR enzyme comprises one or moremutations selected from the group consisting of D917A, E1006A and D1225Aand/or the one or more mutations is in a RuvC domain of the CRISPRenzyme or is a mutation as otherwise as discussed herein. In someembodiments, the CRISPR enzyme has one or more mutations in a catalyticdomain, wherein when transcribed, the direct repeat sequence forms asingle stem loop and the guide sequence directs sequence-specificbinding of a CRISPR complex to the target sequence, and wherein theenzyme further comprises a functional domain. In some embodiments, thefunctional domain is a transcriptional activation domain, preferablyVP64. In some embodiments, the functional domain is a transcriptionrepression domain, preferably KRAB. In some embodiments, thetranscription repression domain is SID, or concatemers of SID (egSID4X). In some embodiments, the functional domain is an epigeneticmodifying domain, such that an epigenetic modifying enzyme is provided.In some embodiments, the functional domain is an activation domain,which may be the P65 activation domain.

Delivery of the C2c1 or C2c3 Effector Protein Complex or ComponentsThereof

Through this disclosure and the knowledge in the art, TALEs, CRISPR-Cassystems, or components thereof or nucleic acid molecules thereof(including, for instance HDR template) or nucleic acid moleculesencoding or providing components thereof may be delivered by a deliverysystem herein described both generally and in detail.

Vector delivery, e.g., plasmid, viral delivery: The CRISPR enzyme, forinstance a Type V protein such as C2c1 or C2c3, and/or any of thepresent RNAs, for instance a guide RNA, can be delivered using anysuitable vector, e.g., plasmid or viral vectors, such as adenoassociated virus (AAV), lentivirus, adenovirus or other viral vectortypes, or combinations thereof. Effector proteins and one or more guideRNAs can be packaged into one or more vectors, e.g., plasmid or viralvectors. In some embodiments, the vector, e.g., plasmid or viral vectoris delivered to the tissue of interest by, for example, an intramuscularinjection, while other times the delivery is via intravenous,transdermal, intranasal, oral, mucosal, or other delivery methods. Suchdelivery may be either via a single dose, or multiple doses. One skilledin the art understands that the actual dosage to be delivered herein mayvary greatly depending upon a variety of factors, such as the vectorchoice, the target cell, organism, or tissue, the general condition ofthe subject to be treated, the degree of transformation/modificationsought, the administration route, the administration mode, the type oftransformation/modification sought, etc.

Such a dosage may further contain, for example, a carrier (water,saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin,dextran, agar, pectin, peanut oil, sesame oil, etc.), a diluent, apharmaceutically-acceptable carrier (e.g., phosphate-buffered saline), apharmaceutically-acceptable excipient, and/or other compounds known inthe art. The dosage may further contain one or more pharmaceuticallyacceptable salts such as, for example, a mineral acid salt such as ahydrochloride, a hydrobromide, a phosphate, a sulfate, etc.; and thesalts of organic acids such as acetates, propionates, malonates,benzoates, etc. Additionally, auxiliary substances, such as wetting oremulsifying agents, pH buffering substances, gels or gelling materials,flavorings, colorants, microspheres, polymers, suspension agents, etc.may also be present herein. In addition, one or more other conventionalpharmaceutical ingredients, such as preservatives, humectants,suspending agents, surfactants, antioxidants, anticaking agents,fillers, chelating agents, coating agents, chemical stabilizers, etc.may also be present, especially if the dosage form is a reconstitutableform. Suitable exemplary ingredients include microcrystalline cellulose,carboxymethylcellulose sodium, polysorbate 80, phenylethyl alcohol,chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propylgallate, the parabens, ethyl vanillin, glycerin, phenol,parachlorophenol, gelatin, albumin and a combination thereof. A thoroughdiscussion of pharmaceutically acceptable excipients is available inREMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991) which isincorporated by reference herein.

In an embodiment herein the delivery is via an adenovirus, which may beat a single booster dose containing at least 1×10⁵ particles (alsoreferred to as particle units, pu) of adenoviral vector. In anembodiment herein, the dose preferably is at least about 1×10⁶ particles(for example, about 1×10⁶-1×10¹¹ particles), more preferably at leastabout 1×10⁷ particles, more preferably at least about 1×10⁸ particles(e.g., about 1×10⁸-1×10¹¹ particles or about 1×10⁹-1×10¹² particles),and most preferably at least about 1×10⁰ particles (e.g., about1×10⁹-1×10¹⁰ particles or about 1×10⁹-1×10¹² particles), or even atleast about 1×10¹⁰ particles (e.g., about 1×10¹⁰-1×10¹² particles) ofthe adenoviral vector. Alternatively, the dose comprises no more thanabout 1×10¹⁴ particles, preferably no more than about 1×10¹³ particles,even more preferably no more than about 1×10¹² particles, even morepreferably no more than about 1×10¹¹ particles, and most preferably nomore than about 1×10¹⁰ particles (e.g., no more than about 1×10⁹articles). Thus, the dose may contain a single dose of adenoviral vectorwith, for example, about 1×10⁶ particle units (pu), about 2×10 ⁶ pu,about 4×10 ⁶ pu, about 1×10⁷ pu, about 2×10 ⁷ pu, about 4×10 ⁷ pu, about1×10⁸ pu, about 2×10 ⁸ pu, about 4×10 ⁸ pu, about 1×10⁹ pu, about 2×10 ⁹pu, about 4×10 ⁹ pu, about 1×10¹⁰ pu, about 2×10 ¹⁰ pu, about 4×10 ¹⁰pu, about 1×10¹¹ pu, about 2×10 ¹¹ pu, about 4×10 ¹¹ pu, about 1×10 ¹²pu, about 2×10 ¹² pu, or about 4×10 ¹² pu of adenoviral vector. See, forexample, the adenoviral vectors in U.S. Pat. No. 8,454,972 B2 to Nabel,et. al., granted on Jun. 4, 2013; incorporated by reference herein, andthe dosages at col 29, lines 36-58 thereof. In an embodiment herein, theadenovirus is delivered via multiple doses.

In an embodiment herein, the delivery is via an AAV. A therapeuticallyeffective dosage for in vivo delivery of the AAV to a human is believedto be in the range of from about 20 to about 50 ml of saline solutioncontaining from about 1×10¹⁰ to about 1×10¹⁰ functional AAV/ml solution.The dosage may be adjusted to balance the therapeutic benefit againstany side effects. In an embodiment herein, the AAV dose is generally inthe range of concentrations of from about 1×10⁵ to 1×10⁵⁰ genomes AAV,from about 1×10⁸ to 1×10²⁰ genomes AAV, from about 1×10¹⁰ to about1×10¹⁶ genomes, or about 1×10¹¹ to about 1×10¹⁶ genomes AAV. A humandosage may be about 1×10¹³ genomes AAV. Such concentrations may bedelivered in from about 0.001 ml to about 100 ml, about 0.05 to about 50ml, or about 10 to about 25 ml of a carrier solution. Other effectivedosages can be readily established by one of ordinary skill in the artthrough routine trials establishing dose response curves. See, forexample, U.S. Pat. No. 8,404,658 B2 to Hajjar, et al., granted on Mar.26, 2013, at col. 27, lines 45-60.

In an embodiment herein the delivery is via a plasmid. In such plasmidcompositions, the dosage should be a sufficient amount of plasmid toelicit a response. For instance, suitable quantities of plasmid DNA inplasmid compositions can be from about 0.1 to about 2 mg, or from about1 μg to about 10 μg per 70 kg individual. Plasmids of the invention willgenerally comprise (i) a promoter; (ii) a sequence encoding an nucleicacid-targeting CRISPR enzyme, operably linked to said promoter; (iii) aselectable marker; (iv) an origin of replication; and (v) atranscription terminator downstream of and operably linked to (ii). Theplasmid can also encode the RNA components of a CRISPR complex, but oneor more of these may instead be encoded on a different vector.

The doses herein are based on an average 70 kg individual. The frequencyof administration is within the ambit of the medical or veterinarypractitioner (e.g., physician, veterinarian), or scientist skilled inthe art. It is also noted that mice used in experiments are typicallyabout 20 g and from mice experiments one can scale up to a 70 kgindividual.

In some embodiments the RNA molecules of the invention are delivered inliposome or lipofectin formulations and the like and can be prepared bymethods well known to those skilled in the art. Such methods aredescribed, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and5,580,859, which are herein incorporated by reference. Delivery systemsaimed specifically at the enhanced and improved delivery of siRNA intomammalian cells have been developed, (see, for example, Shen et al FEBSLet. 2003, 539:111-114; Xia et al., Nat. Biotech. 2002, 20:1006-1010;Reich et al., Mol. Vision. 2003, 9: 210-216; Sorensen et al., J. Mol.Biol. 2003, 327: 761-766; Lewis et al., Nat. Gen. 2002, 32: 107-108 andSimeoni et al., NAR 2003, 31, 11: 2717-2724) and may be applied to thepresent invention. siRNA has recently been successfully used forinhibition of gene expression in primates (see for example. Tolentino etal., Retina 24(4):660 which may also be applied to the presentinvention.

Indeed, RNA delivery is a useful method of in vivo delivery. It ispossible to deliver nucleic acid-targeting Cas proteinCas9 and guideRNAgRNA (and, for instance, HR repair template) into cells usingliposomes or particles. Thus delivery of the nucleic acid-targeting Casprotein/CRISPR enzyme, such as a CasCas9 and/or delivery of the guideRNAs of the invention may be in RNA form and via microvesicles,liposomes or particles. For example, Cas mRNA and guide RNA can bepackaged into liposomal particles for delivery in vivo. Liposomaltransfection reagents such as lipofectamine from Life Technologies andother reagents on the market can effectively deliver RNA molecules intothe liver.

Means of delivery of RNA also preferred include delivery of RNA viananoparticles (Cho, S., Goldberg, M., Son, S., Xu, Q., Yang, F., Mei,Y., Bogatyrev, S., Langer, R. and Anderson, D., Lipid-like nanoparticlesfor small interfering RNA delivery to endothelial cells, AdvancedFunctional Materials, 19: 3112-3118, 2010) or exosomes (Schroeder, A.,Levins, C., Cortez, C., Langer, R., and Anderson, D., Lipid-basednanotherapeutics for siRNA delivery, Journal of Internal Medicine, 267:9-21, 2010, PMID: 20059641). Indeed, exosomes have been shown to beparticularly useful in delivery siRNA, a system with some parallels tothe RNA-targeting system. For instance, El-Andaloussi S, et al.(“Exosome-mediated delivery of siRNA in vitro and in vivo.” Nat Protoc.2012 December; 7(12):2112-26. doi: 10.1038/nprot.2012.131. Epub 2012Nov. 15.) describe how exosomes are promising tools for drug deliveryacross different biological barriers and can be harnessed for deliveryof siRNA in vitro and in vivo. Their approach is to generate targetedexosomes through transfection of an expression vector, comprising anexosomal protein fused with a peptide ligand. The exosomes are thenpurify and characterized from transfected cell supernatant, then RNA isloaded into the exosomes. Delivery or administration according to theinvention can be performed with exosomes, in particular but not limitedto the brain. Vitamin E (α-tocopherol) may be conjugated with nucleicacid-targeting Cas protein and delivered to the brain along with highdensity lipoprotein (HDL), for example in a similar manner as was doneby Uno et al. (HUMAN GENE THERAPY 22:711-719 (June 2011)) for deliveringshort-interfering RNA (siRNA) to the brain. Mice were infused viaOsmotic minipumps (model 1007D; Alzet, Cupertino, Calif.) filled withphosphate-buffered saline (PBS) or free TocsiBACE or Toc-siBACE/HDL andconnected with Brain Infusion Kit 3 (Alzet). A brain-infusion cannulawas placed about 0.5 mm posterior to the bregma at midline for infusioninto the dorsal third ventricle. Uno et al. found that as little as 3nmol of Toc-siRNA with HDL could induce a target reduction in comparabledegree by the same ICV infusion method. A similar dosage of nucleicacid-targeting effector protein conjugated to α-tocopherol andco-administered with HDL targeted to the brain may be contemplated forhumans in the present invention, for example, about 3 nmol to about 3μmol of nucleic acid-targeting effector protein targeted to the brainmay be contemplated. Zou et al. ((HUMAN GENE THERAPY 22:465-475 (April2011)) describes a method of lentiviral-mediated delivery ofshort-hairpin RNAs targeting PKC7 for in vivo gene silencing in thespinal cord of rats. Zou et al. administered about 10 μl of arecombinant lentivirus having a titer of 1×10⁹ transducing units (TU)/mlby an intrathecal catheter. A similar dosage of nucleic acid-targetingeffector protein expressed in a lentiviral vector targeted to the brainmay be contemplated for humans in the present invention, for example,about 10-50 ml of nucleic acid-targeting effector protein targeted tothe brain in a lentivirus having a titer of 1×10⁹ transducing units(TU)/ml may be contemplated.

In terms of local delivery to the brain, this can be achieved in variousways. For instance, material can be delivered intrastriatally e.g., byinjection. Injection can be performed stereotactically via a craniotomy.

Enhancing NHEJ or HR efficiency is also helpful for delivery. It ispreferred that NHEJ efficiency is enhanced by co-expressingend-processing enzymes such as Trex2 (Dumitrache et al. Genetics. 2011August; 188(4): 787-797). It is preferred that HR efficiency isincreased by transiently inhibiting NHEJ machineries such as Ku70 andKu86. HR efficiency can also be increased by co-expressing prokaryoticor eukaryotic homologous recombination enzymes such as RecBCD, RecA.

Packaging and Promoters Generally

Ways to package nucleic acid-targeting effector protein (such as a TypeV protein such as C2c1 or C2c3) coding nucleic acid molecules, e.g.,DNA, into vectors, e.g., viral vectors, to mediate genome modificationin vivo include:

-   -   To achieve NHEJ-mediated gene knockout:    -   Single virus vector:Vector containing two or more expression        cassettes:Promoter-nucleic acid-targeting effector protein        coding nucleic acid molecule-terminatorPromoter-guide        RNA1-terminatorPromoter-guide RNA (N)-terminator (up to size        limit of vector)    -   Double virus vector:Vector 1 containing one expression cassette        for driving the expression of nucleic acid-targeting effector        protein (such as a Type V protein such as C2c1 or        C2c3)Promoter-nucleic acid-targeting effector protein coding        nucleic acid molecule-terminatorVector 2 containing one more        expression cassettes for driving the expression of one or more        guideRNAsPromoter-guide RNA1-terminatorPromoter-guide RNA1        (N)-terminator (up to size limit of vector)    -   To mediate homology-directed repair.    -   In addition to the single and double virus vector approaches        described above, an additional vector is used to deliver a        homology-direct repair template.

The promoter used to drive nucleic acid-targeting effector protein (suchas a Type V protein such as C2c1 or C2c3) coding nucleic acid moleculeexpression can include:

-   -   AAV ITR can serve as a promoter: this is advantageous for        eliminating the need for an additional promoter element (which        can take up space in the vector). The additional space freed up        can be used to drive the expression of additional elements        (gRNA, etc.). Also, ITR activity is relatively weaker, so can be        used to reduce potential toxicity due to over expression of        nucleic acid-targeting effector protein (such as a Type V        protein such as C2c1 or C2c3).    -   For ubiquitous expression, can use promoters: CMV, CAG, CBh,        PGK, SV40, Ferritin heavy or light chains, etc.    -   For brain or other CNS expression, can use promoters: SynapsinI        for all neurons, CaMKIIalpha for excitatory neurons, GAD67 or        GAD65 or VGAT for GABAergic neurons, etc.    -   For liver expression, can use Albumin promoter.    -   For lung expression, can use SP-B.    -   For endothelial cells, can use ICAM.    -   For hematopoietic cells can use IFNbeta or CD45.    -   For Osteoblasts can use OG-2.

The promoter used to drive guide RNA can include:

-   -   Pol III promoters such as U6 or H1    -   Use of Pol II promoter and intronic cassettes to express guide        RNA        Adeno Associated Virus (AAV)

Nucleic acid-targeting effector protein (such as a Type V protein suchas C2c1 or C2c3) and one or more guide RNA can be delivered using adenoassociated virus (AAV), lentivirus, adenovirus or other plasmid or viralvector types, in particular, using formulations and doses from, forexample, U.S. Pat. No. 8,454,972 (formulations, doses for adenovirus),U.S. Pat. No. 8,404,658 (formulations, doses for AAV) and U.S. Pat. No.5,846,946 (formulations, doses for DNA plasmids) and from clinicaltrials and publications regarding the clinical trials involvinglentivirus, AAV and adenovirus. For examples, for AAV, the route ofadministration, formulation and dose can be as in U.S. Pat. No.8,454,972 and as in clinical trials involving AAV. For Adenovirus, theroute of administration, formulation and dose can be as in U.S. Pat. No.8,404,658 and as in clinical trials involving adenovirus. For plasmiddelivery, the route of administration, formulation and dose can be as inU.S. Pat. No. 5,846,946 and as in clinical studies involving plasmids.Doses may be based on or extrapolated to an average 70 kg individual(e.g., a male adult human), and can be adjusted for patients, subjects,mammals of different weight and species. Frequency of administration iswithin the ambit of the medical or veterinary practitioner (e.g.,physician, veterinarian), depending on usual factors including the age,sex, general health, other conditions of the patient or subject and theparticular condition or symptoms being addressed. The viral vectors canbe injected into the tissue of interest. For cell-type specific genomemodification, the expression of nucleic acid-targeting effector protein(such as a Type V protein such as C2c1 or C2c3) can be driven by acell-type specific promoter. For example, liver-specific expressionmight use the Albumin promoter and neuron-specific expression (e.g., fortargeting CNS disorders) might use the Synapsin I promoter.

In terms of in vivo delivery, AAV is advantageous over other viralvectors for a couple of reasons:

-   -   Low toxicity (this may be due to the purification method not        requiring ultra centrifugation of cell particles that can        activate the immune response) and    -   Low probability of causing insertional mutagenesis because it        doesn't integrate into the host genome.

AAV has a packaging limit of 4.5 or 4.75 Kb. This means that nucleicacid-targeting effector protein (such as a Type V protein such as C2c1or C2c3) as well as a promoter and transcription terminator have to beall fit into the same viral vector. Therefore embodiments of theinvention include utilizing homologs of nucleic acid-targeting effectorprotein (such as a Type V protein such as C2c1 or C2c3) that areshorter.

As to AAV, the AAV can be AAV1, AAV2, AAV5 or any combination thereof.One can select the AAV of the AAV with regard to the cells to betargeted; e.g., one can select AAV serotypes 1, 2, 5 or a hybrid capsidAAV1, AAV2, AAV5 or any combination thereof for targeting brain orneuronal cells; and one can select AAV4 for targeting cardiac tissue.AAV8 is useful for delivery to the liver. The herein promoters andvectors are preferred individually. A tabulation of certain AAVserotypes as to these cells (see Grimm, D. et al, J. Virol. 82:5887-5911 (2008)) is as follows:

TABLE 1 Cell Line AAV-1 AAV-2 AAV-3 AAV-4 AAV-5 AAV-6 AAV-8 AAV-9 Huh-713 100 2.5 0.0 0.1 10 0.7 0.0 HEK293 25 100 2.5 0.1 0.1 5 0.7 0.1 HeLa 3100 2.0 0.1 6.7 1 0.2 0.1 HepG2 3 100 16.7 0.3 1.7 5 0.3 ND Hep1A 20 1000.2 1.0 0.1 1 0.2 0.0 911 17 100 11 0.2 0.1 17 0.1 ND CHO 100 100 14 1.4333 50 10   1.0 COS 33 100 33 3.3 5.0 14 2.0 0.5 MeWo 10 100 20 0.3 6.710 1.0 0.2 NIH3T3 10 100 2.9 2.9 0.3 10 0.3 ND A549 14 100 20 ND 0.5 100.5 0.1 HT1180 20 100 10 0.1 0.3 33 0.5 0.1 Monocytes 1111 100 ND ND 1251429 ND ND Immature DC 2500 100 ND ND 222 2857 ND ND Mature DC 2222 100ND ND 333 3333 ND NDLentivirus

Lentiviruses are complex retroviruses that have the ability to infectand express their genes in both mitotic and post-mitotic cells. The mostcommonly known lentivirus is the human immunodeficiency virus (HIV),which uses the envelope glycoproteins of other viruses to target a broadrange of cell types.

Lentiviruses may be prepared as follows. After cloning pCasES10 (whichcontains a lentiviral transfer plasmid backbone), HEK293FT at lowpassage (p=5) were seeded in a T-75 flask to 50% confluence the daybefore transfection in DMEM with 10% fetal bovine serum and withoutantibiotics. After 20 hours, media was changed to OptiMEM (serum-free)media and transfection was done 4 hours later. Cells were transfectedwith 10 μg of lentiviral transfer plasmid (pCasES10) and the followingpackaging plasmids: 5 μg of pMD2.G (VSV-g pseudotype), and 7.5 ug ofpsPAX2 (gag/pol/rev/tat). Transfection was done in 4 mL OptiMEM with acationic lipid delivery agent (50 uL Lipofectamine 2000 and 100 ul Plusreagent). After 6 hours, the media was changed to antibiotic-free DMEMwith 10% fetal bovine serum. These methods use serum during cellculture, but serum-free methods are preferred.

Lentivirus may be purified as follows. Viral supernatants were harvestedafter 48 hours. Supernatants were first cleared of debris and filteredthrough a 0.45 um low protein binding (PVDF) filter. They were then spunin a ultracentrifuge for 2 hours at 24,000 rpm. Viral pellets wereresuspended in 50 ul of DMEM overnight at 4C. They were then aliquotedand immediately frozen at −80° C.

In another embodiment, minimal non-primate lentiviral vectors based onthe equine infectious anemia virus (EIAV) are also contemplated,especially for ocular gene therapy (see, e.g., Balagaan, J Gene Med2006; 8: 275-285). In another embodiment, RetinoStat®, an equineinfectious anemia virus-based lentiviral gene therapy vector thatexpresses angiostatic proteins endostatin and angiostatin that isdelivered via a subretinal injection for the treatment of the web formof age-related macular degeneration is also contemplated (see, e.g.,Binley et al., HUMAN GENE THERAPY 23:980-991 (September 2012)) and thisvector may be modified for the nucleic acid-targeting system of thepresent invention.

In another embodiment, self-inactivating lentiviral vectors with ansiRNA targeting a common exon shared by HIV tat/rev, anucleolar-localizing TAR decoy, and an anti-CCR5-specific hammerheadribozyme (see, e.g., DiGiusto et al. (2010) Sci Transl Med 2:36ra43) maybe used and/or adapted to the nucleic acid-targeting system of thepresent invention. A minimum of 2.5×10⁶ CD34+ cells per kilogram patientweight may be collected and prestimulated for 16 to 20 hours in X-VIVO15 medium (Lonza) containing 2 μmol/L-glutamine, stem cell factor (100ng/ml), Flt-3 ligand (Flt-3L) (100 ng/ml), and thrombopoietin (10 ng/ml)(CellGenix) at a density of 2×10⁶ cells/ml. Prestimulated cells may betransduced with lentiviral at a multiplicity of infection of 5 for 16 to24 hours in 75-cm² tissue culture flasks coated with fibronectin (25mg/cm²) (RetroNectin, Takara Bio Inc.).

Lentiviral vectors have been disclosed as in the treatment forParkinson's Disease, see, e.g., US Patent Publication No. 20120295960and U.S. Pat. Nos. 7,303,910 and 7,351,585. Lentiviral vectors have alsobeen disclosed for the treatment of ocular diseases, see e.g., US PatentPublication Nos. 20060281180, 20090007284, US20110117189; US20090017543;US20070054961, US20100317109. Lentiviral vectors have also beendisclosed for delivery to the brain, see, e.g., US Patent PublicationNos. US20110293571; US20110293571, US20040013648, US20070025970,US20090111106 and U.S. Pat. No. 7,259,015. RNA Delivery

RNA delivery: The nucleic acid-targeting Cas protein, for instance aType V protein such as C2c1 or C2c3, and/or guide RNA, can also bedelivered in the form of RNA. nucleic acid-targeting Cas protein (suchas a Type V protein such as C2c1 or C2c3) mRNA can be generated using invitro transcription. For example, nucleic acid-targeting effectorprotein (such as a Type V protein such as C2c1 or C2c3) mRNA can besynthesized using a PCR cassette containing the following elements:T7_promoter-kozak sequence (GCCACC)-effector protrein-3′ UTR from betaglobin-polyA tail (a string of 120 or more adenines). The cassette canbe used for transcription by T7 polymerase. Guide RNAs can also betranscribed using in vitro transcription from a cassette containingT7_promoter-GG-guide RNA sequence.

To enhance expression and reduce possible toxicity, the nucleicacid-targeting effector protein-coding sequence and/or the guide RNA canbe modified to include one or more modified nucleoside e.g., usingpseudo-U or 5-Methyl-C.

mRNA delivery methods are especially promising for liver deliverycurrently.

Much clinical work on RNA delivery has focused on RNAi or antisense, butthese systems can be adapted for delivery of RNA for implementing thepresent invention. References below to RNAi etc. should be readaccordingly.

Particle Delivery Systems and/or Formulations:

Several types of particle delivery systems and/or formulations are knownto be useful in a diverse spectrum of biomedical applications. Ingeneral, a particle is defined as a small object that behaves as a wholeunit with respect to its transport and properties. Particles are furtherclassified according to diameter. Coarse particles cover a range between2,500 and 10,000 nanometers. Fine particles are sized between 100 and2,500 nanometers. Ultrafine particles, or nanoparticles, are generallybetween 1 and 100 nanometers in size. The basis of the 100-nm limit isthe fact that novel properties that differentiate particles from thebulk material typically develop at a critical length scale of under 100nm.

As used herein, a particle delivery system/formulation is defined as anybiological delivery system/formulation which includes a particle inaccordance with the present invention. A particle in accordance with thepresent invention is any entity having a greatest dimension (e.g.diameter) of less than 100 microns (pm). In some embodiments, inventiveparticles have a greatest dimension of less than 10 pm. In someembodiments, inventive particles have a greatest dimension of less than2000 nanometers (nm). In some embodiments, inventive particles have agreatest dimension of less than 1000 nanometers (nm). In someembodiments, inventive particles have a greatest dimension of less than900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, or 100nm. Typically, inventive particles have a greatest dimension (e.g.,diameter) of 500 nm or less. In some embodiments, inventive particleshave a greatest dimension (e.g., diameter) of 250 nm or less. In someembodiments, inventive particles have a greatest dimension (e.g.,diameter) of 200 nm or less. In some embodiments, inventive particleshave a greatest dimension (e.g., diameter) of 150 nm or less. In someembodiments, inventive particles have a greatest dimension (e.g.,diameter) of 100 nm or less. Smaller particles, e.g., having a greatestdimension of 50 nm or less are used in some embodiments of theinvention. In some embodiments, inventive particles have a greatestdimension ranging between 25 nm and 200 nm.

Particle characterization (including e.g., characterizing morphology,dimension, etc.) is done using a variety of different techniques. Commontechniques are electron microscopy (TEM, SEM), atomic force microscopy(AFM), dynamic light scattering (DLS), X-ray photoelectron spectroscopy(XPS), powder X-ray diffraction (XRD), Fourier transform infraredspectroscopy (FTIR), matrix-assisted laser desorption/onizationtime-of-flight mass spectrometry (MALDI-TOF), ultraviolet-visiblespectroscopy, dual polarisation interferometry and nuclear magneticresonance (NMR). Characterization (dimension measurements) may be madeas to native particles (i.e., preloading) or after loading of the cargo(herein cargo refers to e.g., one or more components of CRISPR-Cassystem e.g., CRISPR enzyme or mRNA or guide RNA, or any combinationthereof, and may include additional carriers and/or excipients) toprovide particles of an optimal size for delivery for any in vitro, exvivo and/or in vivo application of the present invention. In certainpreferred embodiments, particle dimension (e.g., diameter)characterization is based on measurements using dynamic laser scattering(DLS). Mention is made of U.S. Pat. Nos. 8,709,843; 6,007,845;5,855,913; 5,985,309; 5,543,158; and the publication by James E. Dahlmanand Carmen Barnes et al. Nature Nanotechnology (2014) published online11 May 2014, doi:10.1038/nnano.2014.84, concerning particles, methods ofmaking and using them and measurements thereof.

Particles delivery systems within the scope of the present invention maybe provided in any form, including but not limited to solid, semi-solid,emulsion, or colloidal particles. As such any of the delivery systemsdescribed herein, including but not limited to, e.g., lipid-basedsystems, liposomes, micelles, microvesicles, exosomes, or gene gun maybe provided as particle delivery systems within the scope of the presentinvention.

Particles

CRISPR enzyme mRNA and guide RNA may be delivered simultaneously usingparticles or lipid envelopes; for instance, CRISPR enzyme and RNA of theinvention, e.g., as a complex, can be delivered via a particle as inDahlman et al., WO2015089419 A2 and documents cited therein, such as 7C1(see, e.g., James E. Dahlman and Carmen Barnes et al. NatureNanotechnology (2014) published online 11 May 2014,doi:10.1038/nnano.2014.84), e.g., delivery particle comprising lipid orlipidoid and hydrophilic polymer, e.g., cationic lipid and hydrophilicpolymer, for instance wherein the cationic lipid comprises1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC) and/or whereinthe hydrophilic polymer comprises ethylene glycol or polyethylene glycol(PEG); and/or wherein the particle further comprises cholesterol (e.g.,particle from formulation 1=DOTAP 100, DMPC 0, PEG 0, Cholesterol 0;formulation number 2=DOTAP 90, DMPC 0, PEG 10, Cholesterol 0;formulation number 3=DOTAP 90, DMPC 0, PEG 5, Cholesterol 5), whereinparticles are formed using an efficient, multistep process whereinfirst, effector protein and RNA are mixed together, e.g., at a 1:1 molarratio, e.g., at room temperature, e.g., for 30 minutes, e.g., insterile, nuclease free 1×PBS; and separately, DOTAP, DMPC, PEG, andcholesterol as applicable for the formulation are dissolved in alcohol,e.g., 100% ethanol; and, the two solutions are mixed together to formparticles containing the complexes).

Nucleic acid-targeting effector proteins (such as a Type V protein suchas C2c1 or C2c3) mRNA and guide RNA may be delivered simultaneouslyusing particles or lipid envelopes.

For example, Su X, Fricke J, Kavanagh D G, Irvine D J (“In vitro and invivo mRNA delivery using lipid-enveloped pH-responsive polymernanoparticles” Mol Pharm. 2011 Jun. 6; 8(3):774-87. doi:10.1021/mp100390w. Epub 2011 Apr. 1) describes biodegradable core-shellstructured particles with a poly(β-amino ester) (PBAE) core enveloped bya phospholipid bilayer shell. These were developed for in vivo mRNAdelivery. The pH-responsive PBAE component was chosen to promoteendosome disruption, while the lipid surface layer was selected tominimize toxicity of the polycation core. Such are, therefore, preferredfor delivering RNA of the present invention.

In one embodiment, particles based on self-assembling bioadhesivepolymers are contemplated, which may be applied to oral delivery ofpeptides, intravenous delivery of peptides and nasal delivery ofpeptides, all to the brain. Other embodiments, such as oral absorptionand ocular delivery of hydrophobic drugs are also contemplated. Themolecular envelope technology involves an engineered polymer envelopewhich is protected and delivered to the site of the disease (see, e.g.,Mazza, M. et al. ACS Nano, 2013. 7(2): 1016-1026; Siew, A., et al. MolPharm, 2012. 9(1):14-28; Lalatsa, A., et al. J Contr Rel, 2012.161(2):523-36; Lalatsa, A., et al., Mol Pharm, 2012. 9(6):1665-80;Lalatsa, A., et al. Mol Pharm, 2012. 9(6):1764-74; Garrett, N. L., etal. J Biophotonics, 2012. 5(5-6):458-68; Garrett, N. L., et al. J RamanSpect, 2012. 43(5):681-688; Ahmad, S., et al. J Royal Soc Interface2010. 7:S423-33; Uchegbu, I. F. Expert Opin Drug Deliv, 2006.3(5):629-40; Qu, X., et al. Biomacromolecules, 2006. 7(12):3452-9 andUchegbu, I. F., et al. Int J Pharm, 2001. 224:185-199). Doses of about 5mg/kg are contemplated, with single or multiple doses, depending on thetarget tissue.

In one embodiment, particles that can deliver RNA to a cancer cell tostop tumor growth developed by Dan Anderson's lab at MIT may be usedand/or adapted to the nucleic acid-targeting system of the presentinvention. In particular, the Anderson lab developed fully automated,combinatorial systems for the synthesis, purification, characterization,and formulation of new biomaterials and nanoformulations. See, e.g.,Alabi et al., Proc Natl Acad Sci USA. 2013 Aug. 6; 110(32):12881-6;Zhang et al., Adv Mater. 2013 Sep. 6; 25(33):4641-5; Jiang et al., NanoLett. 2013 Mar. 13; 13(3):1059-64; Karagiannis et al., ACS Nano. 2012Oct. 23; 6(10):8484-7; Whitehead et al., ACS Nano. 2012 Aug. 28;6(8):6922-9 and Lee et al., Nat Nanotechnol. 2012 Jun. 3; 7(6):389-93.

US patent application 20110293703 relates to lipidoid compounds are alsoparticularly useful in the administration of polynucleotides, which maybe applied to deliver the nucleic acid-targeting system of the presentinvention. In one aspect, the aminoalcohol lipidoid compounds arecombined with an agent to be delivered to a cell or a subject to formmicroparticles, nanoparticles, liposomes, or micelles. The agent to bedelivered by the particles, liposomes, or micelles may be in the form ofa gas, liquid, or solid, and the agent may be a polynucleotide, protein,peptide, or small molecule. The minoalcohol lipidoid compounds may becombined with other aminoalcohol lipidoid compounds, polymers (syntheticor natural), surfactants, cholesterol, carbohydrates, proteins, lipids,etc. to form the particles. These particles may then optionally becombined with a pharmaceutical excipient to form a pharmaceuticalcomposition.

US Patent Publication No. 20110293703 also provides methods of preparingthe aminoalcohol lipidoid compounds. One or more equivalents of an amineare allowed to react with one or more equivalents of anepoxide-terminated compound under suitable conditions to form anaminoalcohol lipidoid compound of the present invention. In certainembodiments, all the amino groups of the amine are fully reacted withthe epoxide-terminated compound to form tertiary amines. In otherembodiments, all the amino groups of the amine are not fully reactedwith the epoxide-terminated compound to form tertiary amines therebyresulting in primary or secondary amines in the aminoalcohol lipidoidcompound. These primary or secondary amines are left as is or may bereacted with another electrophile such as a different epoxide-terminatedcompound. As will be appreciated by one skilled in the art, reacting anamine with less than excess of epoxide-terminated compound will resultin a plurality of different aminoalcohol lipidoid compounds with variousnumbers of tails. Certain amines may be fully functionalized with twoepoxide-derived compound tails while other molecules will not becompletely functionalized with epoxide-derived compound tails. Forexample, a diamine or polyamine may include one, two, three, or fourepoxide-derived compound tails off the various amino moieties of themolecule resulting in primary, secondary, and tertiary amines. Incertain embodiments, all the amino groups are not fully functionalized.In certain embodiments, two of the same types of epoxide-terminatedcompounds are used. In other embodiments, two or more differentepoxide-terminated compounds are used. The synthesis of the aminoalcohollipidoid compounds is performed with or without solvent, and thesynthesis may be performed at higher temperatures ranging from 30-100°C., preferably at approximately 50-90° C. The prepared aminoalcohollipidoid compounds may be optionally purified. For example, the mixtureof aminoalcohol lipidoid compounds may be purified to yield anaminoalcohol lipidoid compound with a particular number ofepoxide-derived compound tails. Or the mixture may be purified to yielda particular stereo- or regioisomer. The aminoalcohol lipidoid compoundsmay also be alkylated using an alkyl halide (e.g., methyl iodide) orother alkylating agent, and/or they may be acylated.

US Patent Publication No. 20110293703 also provides libraries ofaminoalcohol lipidoid compounds prepared by the inventive methods. Theseaminoalcohol lipidoid compounds may be prepared and/or screened usinghigh-throughput techniques involving liquid handlers, robots, microtiterplates, computers, etc. In certain embodiments, the aminoalcohollipidoid compounds are screened for their ability to transfectpolynucleotides or other agents (e.g., proteins, peptides, smallmolecules) into the cell.

US Patent Publication No. 20130302401 relates to a class ofpoly(beta-amino alcohols) (PBAAs) has been prepared using combinatorialpolymerization. The inventive PBAAs may be used in biotechnology andbiomedical applications as coatings (such as coatings of films ormultilayer films for medical devices or implants), additives, materials,excipients, non-biofouling agents, micropatterning agents, and cellularencapsulation agents. When used as surface coatings, these PBAAselicited different levels of inflammation, both in vitro and in vivo,depending on their chemical structures. The large chemical diversity ofthis class of materials allowed us to identify polymer coatings thatinhibit macrophage activation in vitro. Furthermore, these coatingsreduce the recruitment of inflammatory cells, and reduce fibrosis,following the subcutaneous implantation of carboxylated polystyrenemicroparticles. These polymers may be used to form polyelectrolytecomplex capsules for cell encapsulation. The invention may also havemany other biological applications such as antimicrobial coatings, DNAor siRNA delivery, and stem cell tissue engineering. The teachings of USPatent Publication No. 20130302401 may be applied to the nucleicacid-targeting system of the present invention.

In another embodiment, lipid nanoparticles (LNPs) are contemplated. Anantitransthyretin small interfering RNA has been encapsulated in lipidnanoparticles and delivered to humans (see, e.g., Coelho et al., N.Engl. J. Med 2013; 369:819-29), and such a system may be adapted andapplied to the nucleic acid-targeting system of the present invention.Doses of about 0.01 to about 1 mg per kg of body weight administeredintravenously are contemplated. Medications to reduce the risk ofinfusion-related reactions are contemplated, such as dexamethasone,acetaminophen, diphenhydramine or cetirizine, and ranitidine arecontemplated. Multiple doses of about 0.3 mg per kilogram every 4 weeksfor five doses are also contemplated.

LNPs have been shown to be highly effective in delivering siRNAs to theliver (see, e.g., Tabernero et al., Cancer Discovery, April 2013, Vol.3, No. 4, pages 363-470) and are therefore contemplated for deliveringRNA encoding nucleic acid-targeting effector protein to the liver. Adosage of about four doses of 6 mg/kg of the LNP every two weeks may becontemplated. Tabernero et al. demonstrated that tumor regression wasobserved after the first 2 cycles of LNPs dosed at 0.7 mg/kg, and by theend of 6 cycles the patient had achieved a partial response withcomplete regression of the lymph node metastasis and substantialshrinkage of the liver tumors. A complete response was obtained after 40doses in this patient, who has remained in remission and completedtreatment after receiving doses over 26 months. Two patients with RCCand extrahepatic sites of disease including kidney, lung, and lymphnodes that were progressing following prior therapy with VEGF pathwayinhibitors had stable disease at all sites for approximately 8 to 12months, and a patient with PNET and liver metastases continued on theextension study for 18 months (36 doses) with stable disease.

However, the charge of the LNP must be taken into consideration. Ascationic lipids combined with negatively charged lipids to inducenonbilayer structures that facilitate intracellular delivery. Becausecharged LNPs are rapidly cleared from circulation following intravenousinjection, ionizable cationic lipids with pKa values below 7 weredeveloped (see, e.g., Rosin et al, Molecular Therapy, vol. 19, no. 12,pages 1286-2200, December 2011). Negatively charged polymers such as RNAmay be loaded into LNPs at low pH values (e.g., pH 4) where theionizable lipids display a positive charge. However, at physiological pHvalues, the LNPs exhibit a low surface charge compatible with longercirculation times. Four species of ionizable cationic lipids have beenfocused upon, namely 1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP),1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA),1,2-dilinoleyloxy-keto-N,N-dimethyl-3-aminopropane (DLinKDMA), and1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA).It has been shown that LNP siRNA systems containing these lipids exhibitremarkably different gene silencing properties in hepatocytes in vivo,with potencies varying according to the seriesDLinKC2-DMA>DLinKDMA>DLinDMA>>DLinDAP employing a Factor VII genesilencing model (see, e.g., Rosin et al, Molecular Therapy, vol. 19, no.12, pages 1286-2200, December 2011). A dosage of 1 μg/ml of LNP orCRISPR-Cas RNA in or associated with the LNP may be contemplated,especially for a formulation containing DLinKC2-DMA.

Preparation of LNPs and CRISPR-Cas encapsulation may be used and/oradapted from Rosin et al, Molecular Therapy, vol. 19, no. 12, pages1286-2200, December 2011). The cationic lipids1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP),1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA),1,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA),1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA),(3-o-[2″-(methoxypolyethyleneglycol 2000)succinoyl]-1,2-dimyristoyl-sn-glycol (PEG-S-DMG), andR-3-[(o-methoxy-poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxylpropyl-3-amine (PEG-C-DOMG) may be providedby Tekmira Pharmaceuticals (Vancouver, Canada) or synthesized.Cholesterol may be purchased from Sigma (St Louis, Mo.). The specificnucleic acid-targeting complex (CRISPR-Cas) RNA may be encapsulated inLNPs containing DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA (cationiclipid:DSPC:CHOL: PEGS-DMG or PEG-C-DOMG at 40:10:40:10 molar ratios).When required, 0.2% SP-DiOC18 (Invitrogen, Burlington, Canada) may beincorporated to assess cellular uptake, intracellular delivery, andbiodistribution. Encapsulation may be performed by dissolving lipidmixtures comprised of cationic lipid:DSPC:cholesterol:PEG-c-DOMG(40:10:40:10 molar ratio) in ethanol to a final lipid concentration of10 mmol/l. This ethanol solution of lipid may be added drop-wise to 50mmol/l citrate, pH 4.0 to form multilamellar vesicles to produce a finalconcentration of 30% ethanol vol/vol. Large unilamellar vesicles may beformed following extrusion of multilamellar vesicles through two stacked80 nm Nuclepore polycarbonate filters using the Extruder (NorthernLipids, Vancouver, Canada). Encapsulation may be achieved by adding RNAdissolved at 2 mg/ml in 50 mmol/l citrate, pH 4.0 containing 30% ethanolvol/vol drop-wise to extruded preformed large unilamellar vesicles andincubation at 31° C. for 30 minutes with constant mixing to a finalRNA/lipid weight ratio of 0.06/1 wt/wt. Removal of ethanol andneutralization of formulation buffer were performed by dialysis againstphosphate-buffered saline (PBS), pH 7.4 for 16 hours using Spectra/Por 2regenerated cellulose dialysis membranes. Particle size distribution maybe determined by dynamic light scattering using a NICOMP 370 particlesizer, the vesicle/intensity modes, and Gaussian fitting (NicompParticle Sizing, Santa Barbara, Calif.). The particle size for all threeLNP systems may be −70 nm in diameter. RNA encapsulation efficiency maybe determined by removal of free RNA using VivaPureD MiniH columns(Sartorius Stedim Biotech) from samples collected before and afterdialysis. The encapsulated RNA may be extracted from the elutedparticles and quantified at 260 nm. RNA to lipid ratio was determined bymeasurement of cholesterol content in vesicles using the Cholesterol Eenzymatic assay from Wako Chemicals USA (Richmond, Va.). In conjunctionwith the herein discussion of LNPs and PEG lipids, PEGylated liposomesor LNPs are likewise suitable for delivery of a nucleic acid-targetingsystem or components thereof.

Preparation of large LNPs may be used and/or adapted from Rosin et al,Molecular Therapy, vol. 19, no. 12, pages 1286-2200, December 2011. Alipid premix solution (20.4 mg/ml total lipid concentration) may beprepared in ethanol containing DLinKC2-DMA, DSPC, and cholesterol at50:10:38.5 molar ratios. Sodium acetate may be added to the lipid premixat a molar ratio of 0.75:1 (sodium acetate:DLinKC2-DMA). The lipids maybe subsequently hydrated by combining the mixture with 1.85 volumes ofcitrate buffer (10 mmol/l, pH 3.0) with vigorous stirring, resulting inspontaneous liposome formation in aqueous buffer containing 35% ethanol.The liposome solution may be incubated at 37° C. to allow fortime-dependent increase in particle size. Aliquots may be removed atvarious times during incubation to investigate changes in liposome sizeby dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments,Worcestershire, UK). Once the desired particle size is achieved, anaqueous PEG lipid solution (stock=10 mg/ml PEG-DMG in 35% (vol/vol)ethanol) may be added to the liposome mixture to yield a final PEG molarconcentration of 3.5% of total lipid. Upon addition of PEG-lipids, theliposomes should their size, effectively quenching further growth. RNAmay then be added to the empty liposomes at a RNA to total lipid ratioof approximately 1:10 (wt:wt), followed by incubation for 30 minutes at37° C. to form loaded LNPs. The mixture may be subsequently dialyzedovernight in PBS and filtered with a 0.45-μm syringe filter.

Spherical Nucleic Acid (SNA™) constructs and other particles(particularly gold particles) are also contemplated as a means todelivery nucleic acid-targeting system to intended targets. Significantdata show that AuraSense Therapeutics' Spherical Nucleic Acid (SNA™)constructs, based upon nucleic acid-functionalized gold particles, areuseful.

Literature that may be employed in conjunction with herein teachingsinclude: Cutler et al., J. Am. Chem. Soc. 2011 133:9254-9257, Hao etal., Small. 2011 7:3158-3162, Zhang et al., ACS Nano. 2011 5:6962-6970,Cutler et al., J. Am. Chem. Soc. 2012 134:1376-1391, Young et al., NanoLett. 2012 12:3867-71, Zheng et al., Proc. Natl. Acad. Sci. USA. 2012109:11975-80, Mirkin, Nanomedicine 2012 7:635-638 Zhang et al., J. Am.Chem. Soc. 2012 134:16488-1691, Weintraub, Nature 2013 495:S14-S16, Choiet al., Proc. Natl. Acad. Sci. USA. 2013 110(19):7625-7630, Jensen etal., Sci. Transl. Med 5, 209ra152 (2013) and Mirkin, et al., Small,10:186-192.

Self-assembling particles with RNA may be constructed withpolyethyleneimine (PEI) that is PEGylated with an Arg-Gly-Asp (RGD)peptide ligand attached at the distal end of the polyethylene glycol(PEG). This system has been used, for example, as a means to targettumor neovasculature expressing integrins and deliver siRNA inhibitingvascular endothelial growth factor receptor-2 (VEGF R2) expression andthereby achieve tumor angiogenesis (see, e.g., Schiffelers et al.,Nucleic Acids Research, 2004, Vol. 32, No. 19). Nanoplexes may beprepared by mixing equal volumes of aqueous solutions of cationicpolymer and nucleic acid to give a net molar excess of ionizablenitrogen (polymer) to phosphate (nucleic acid) over the range of 2 to 6.The electrostatic interactions between cationic polymers and nucleicacid resulted in the formation of polyplexes with average particle sizedistribution of about 100 nm, hence referred to here as nanoplexes. Adosage of about 100 to 200 mg of nucleic acid-targeting complex RNA isenvisioned for delivery in the self-assembling particles of Schiffelerset al.

The nanoplexes of Bartlett et al. (PNAS, Sep. 25, 2007, vol. 104, no.39) may also be applied to the present invention. The nanoplexes ofBartlett et al. are prepared by mixing equal volumes of aqueoussolutions of cationic polymer and nucleic acid to give a net molarexcess of ionizable nitrogen (polymer) to phosphate (nucleic acid) overthe range of 2 to 6. The electrostatic interactions between cationicpolymers and nucleic acid resulted in the formation of polyplexes withaverage particle size distribution of about 100 nm, hence referred tohere as nanoplexes. The DOTA-siRNA of Bartlett et al. was synthesized asfollows: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acidmono(N-hydroxysuccinimide ester) (DOTA-NHSester) was ordered fromMacrocyclics (Dallas, Tex.). The amine modified RNA sense strand with a100-fold molar excess of DOTA-NHS-ester in carbonate buffer (pH 9) wasadded to a microcentrifuge tube. The contents were reacted by stirringfor 4 h at room temperature. The DOTA-RNAsense conjugate wasethanol-precipitated, resuspended in water, and annealed to theunmodified antisense strand to yield DOTA-siRNA. All liquids werepretreated with Chelex-100 (Bio-Rad, Hercules, Calif.) to remove tracemetal contaminants. Tf-targeted and nontargeted siRNA particles may beformed by using cyclodextrin-containing polycations. Typically,particles were formed in water at a charge ratio of 3 (+/−) and an siRNAconcentration of 0.5 g/liter. One percent of the adamantane-PEGmolecules on the surface of the targeted particles were modified with Tf(adamantane-PEG-Tf). The particles were suspended in a 5% (wt/vol)glucose carrier solution for injection.

Davis et al. (Nature, Vol 464, 15 Apr. 2010) conducts a RNA clinicaltrial that uses a targeted particle-delivery system (clinical trialregistration number NCT00689065). Patients with solid cancers refractoryto standard-of-care therapies are administered doses of targetedparticles on days 1, 3, 8 and 10 of a 21-day cycle by a 30-minintravenous infusion. The particles comprise, consist essentially of, orconsist of a synthetic delivery system containing: (1) a linear,cyclodextrin-based polymer (CDP), (2) a human transferrin protein (TF)targeting ligand displayed on the exterior of the nanoparticle to engageTF receptors (TFR) on the surface of the cancer cells, (3) a hydrophilicpolymer (polyethylene glycol (PEG) used to promote nanoparticlestability in biological fluids), and (4) siRNA designed to reduce theexpression of the RRM2 (sequence used in the clinic was previouslydenoted siR2B+5). The TFR has long been known to be upregulated inmalignant cells, and RRM2 is an established anti-cancer target. Theseparticles (clinical version denoted as CALAA-01) have been shown to bewell tolerated in multi-dosing studies in non-human primates. Although asingle patient with chronic myeloid leukaemia has been administeredsiRNA by liposomal delivery, Davis et al.'s clinical trial is theinitial human trial to systemically deliver siRNA with a targeteddelivery system and to treat patients with solid cancer. To ascertainwhether the targeted delivery system can provide effective delivery offunctional siRNA to human tumours, Davis et al. investigated biopsiesfrom three patients from three different dosing cohorts; patients A, Band C, all of whom had metastatic melanoma and received CALAA-01 dosesof 18, 24 and 30 mg m² siRNA, respectively. Similar doses may also becontemplated for the nucleic acid-targeting system of the presentinvention. The delivery of the invention may be achieved with particlescontaining a linear, cyclodextrin-based polymer (CDP), a humantransferrin protein (TF) targeting ligand displayed on the exterior ofthe particle to engage TF receptors (TFR) on the surface of the cancercells and/or a hydrophilic polymer (for example, polyethylene glycol(PEG) used to promote particle stability in biological fluids).

In terms of this invention, it is preferred to have one or morecomponents of nucleic acid-targeting complex, e.g., nucleicacid-targeting effector protein or mRNA, or guide RNA delivered usingparticles or lipid envelopes. Other delivery systems or vectors are maybe used in conjunction with the particle aspects of the invention.

In general, a “nanoparticle” refers to any particle having a diameter ofless than 1000 nm. In certain preferred embodiments, nanoparticles ofthe invention have a greatest dimension (e.g., diameter) of 500 nm orless. In other preferred embodiments, nanoparticles of the inventionhave a greatest dimension ranging between 25 nm and 200 nm. In otherpreferred embodiments, particles of the invention have a greatestdimension of 100 nm or less. In other preferred embodiments,nanoparticles of the invention have a greatest dimension ranging between35 nm and 60 nm.

Particles encompassed in the present invention may be provided indifferent forms, e.g., as solid particles (e.g., metal such as silver,gold, iron, titanium), non-metal, lipid-based solids, polymers),suspensions of particles, or combinations thereof. Metal, dielectric,and semiconductor particles may be prepared, as well as hybridstructures (e.g., core-shell particles). Particles made ofsemiconducting material may also be labeled quantum dots if they aresmall enough (typically sub 10 nm) that quantization of electronicenergy levels occurs. Such nanoscale particles are used in biomedicalapplications as drug carriers or imaging agents and may be adapted forsimilar purposes in the present invention.

Semi-solid and soft particles have been manufactured, and are within thescope of the present invention. A prototype particle of semi-solidnature is the liposome. Various types of liposome particles arecurrently used clinically as delivery systems for anticancer drugs andvaccines. Particles with one half hydrophilic and the other halfhydrophobic are termed Janus particles and are particularly effectivefor stabilizing emulsions. They can self-assemble at water/oilinterfaces and act as solid surfactants.

U.S. Pat. No. 8,709,843, incorporated herein by reference, provides adrug delivery system for targeted delivery of therapeuticagent-containing particles to tissues, cells, and intracellularcompartments. The invention provides targeted particles comprisingpolymer conjugated to a surfactant, hydrophilic polymer or lipid.

U.S. Pat. No. 6,007,845, incorporated herein by reference, providesparticles which have a core of a multiblock copolymer formed bycovalently linking a multifunctional compound with one or morehydrophobic polymers and one or more hydrophilic polymers, and contain abiologically active material.

U.S. Pat. No. 5,855,913, incorporated herein by reference, provides aparticulate composition having aerodynamically light particles having atap density of less than 0.4 g/cm3 with a mean diameter of between 5 pmand 30 pm, incorporating a surfactant on the surface thereof for drugdelivery to the pulmonary system.

U.S. Pat. No. 5,985,309, incorporated herein by reference, providesparticles incorporating a surfactant and/or a hydrophilic or hydrophobiccomplex of a positively or negatively charged therapeutic or diagnosticagent and a charged molecule of opposite charge for delivery to thepulmonary system.

U.S. Pat. No. 5,543,158, incorporated herein by reference, providesbiodegradable injectable particles having a biodegradable solid corecontaining a biologically active material and poly(alkylene glycol)moieties on the surface.

WO2012135025 (also published as US20120251560), incorporated herein byreference, describes conjugated polyethyleneimine (PEI) polymers andconjugated aza-macrocycles (collectively referred to as “conjugatedlipomer” or “lipomers”). In certain embodiments, it can be envisionedthat such methods and materials of herein-cited documents, e.g.,conjugated lipomers can be used in the context of the nucleicacid-targeting system to achieve in vitro, ex vivo and in vivo genomicperturbations to modify gene expression, including modulation of proteinexpression.

In one embodiment, the particle may be epoxide-modified lipid-polymer,advantageously 7C1 (see, e.g., James E. Dahlman and Carmen Barnes et al.Nature Nanotechnology (2014) published online 11 May 2014,doi:10.1038/nnano.2014.84). C71 was synthesized by reacting C15epoxide-terminated lipids with PEI600 at a 14:1 molar ratio, and wasformulated with C14PEG2000 to produce particles (diameter between 35 and60 nm) that were stable in PBS solution for at least 40 days.

An epoxide-modified lipid-polymer may be utilized to deliver the nucleicacid-targeting system of the present invention to pulmonary,cardiovascular or renal cells, however, one of skill in the art mayadapt the system to deliver to other target organs. Dosage ranging fromabout 0.05 to about 0.6 mg/kg are envisioned. Dosages over several daysor weeks are also envisioned, with a total dosage of about 2 mg/kg.

Exosomes

Exosomes are endogenous nano-vesicles that transport RNAs and proteins,and which can deliver RNA to the brain and other target organs. Toreduce immunogenicity, Alvarez-Erviti et al. (2011, Nat Biotechnol 29:341) used self-derived dendritic cells for exosome production. Targetingto the brain was achieved by engineering the dendritic cells to expressLamp2b, an exosomal membrane protein, fused to the neuron-specific RVGpeptide. Purified exosomes were loaded with exogenous RNA byelectroporation. Intravenously injected RVG-targeted exosomes deliveredGAPDH siRNA specifically to neurons, microglia, oligodendrocytes in thebrain, resulting in a specific gene knockdown. Pre-exposure to RVGexosomes did not attenuate knockdown, and non-specific uptake in othertissues was not observed. The therapeutic potential of exosome-mediatedsiRNA delivery was demonstrated by the strong mRNA (60%) and protein(62%) knockdown of BACE1, a therapeutic target in Alzheimer's disease.

To obtain a pool of immunologically inert exosomes, Alvarez-Erviti etal. harvested bone marrow from inbred C57BL/6 mice with a homogenousmajor histocompatibility complex (MHC) haplotype. As immature dendriticcells produce large quantities of exosomes devoid of T-cell activatorssuch as MHC-II and CD86, Alvarez-Erviti et al. selected for dendriticcells with granulocyte/macrophage-colony stimulating factor (GM-CSF) for7 d. Exosomes were purified from the culture supernatant the followingday using well-established ultracentrifugation protocols. The exosomesproduced were physically homogenous, with a size distribution peaking at80 nm in diameter as determined by particle tracking analysis (NTA) andelectron microscopy. Alvarez-Erviti et al. obtained 6-12 μg of exosomes(measured based on protein concentration) per 10⁶ cells.

Next, Alvarez-Erviti et al. investigated the possibility of loadingmodified exosomes with exogenous cargoes using electroporation protocolsadapted for nanoscale applications. As electroporation for membraneparticles at the nanometer scale is not well-characterized, nonspecificCy5-labeled RNA was used for the empirical optimization of theelectroporation protocol. The amount of encapsulated RNA was assayedafter ultracentrifugation and lysis of exosomes. Electroporation at 400V and 125 pF resulted in the greatest retention of RNA and was used forall subsequent experiments.

Alvarez-Erviti et al. administered 150 μg of each BACE1 siRNAencapsulated in 150 μg of RVG exosomes to normal C57BL/6 mice andcompared the knockdown efficiency to four controls: untreated mice, miceinjected with RVG exosomes only, mice injected with BACE1 siRNAcomplexed to an in vivo cationic liposome reagent and mice injected withBACE1 siRNA complexed to RVG-9R, the RVG peptide conjugated to 9D-arginines that electrostatically binds to the siRNA. Cortical tissuesamples were analyzed 3 d after administration and a significant proteinknockdown (45%, P<0.05, versus 62%, P<0.01) in both siRNA-RVG-9R-treatedand siRNARVG exosome-treated mice was observed, resulting from asignificant decrease in BACE1 mRNA levels (66% [+ or −] 15%, P<0.001 and61% [+ or −] 13% respectively, P<0.01). Moreover, Applicantsdemonstrated a significant decrease (55%, P<0.05) in the total[beta]-amyloid 1-42 levels, a main component of the amyloid plaques inAlzheimer's pathology, in the RVG-exosome-treated animals. The decreaseobserved was greater than the β-amyloid 1-40 decrease demonstrated innormal mice after intraventricular injection of BACE1 inhibitors.Alvarez-Erviti et al. carried out 5′-rapid amplification of cDNA ends(RACE) on BACE1 cleavage product, which provided evidence ofRNAi-mediated knockdown by the siRNA.

Finally, Alvarez-Erviti et al. investigated whether RNA-RVG exosomesinduced immune responses in vivo by assessing IL-6, IP-10, TNFα andIFN-α serum concentrations. Following exosome treatment, nonsignificantchanges in all cytokines were registered similar to siRNA-transfectionreagent treatment in contrast to siRNA-RVG-9R, which potently stimulatedIL-6 secretion, confirming the immunologically inert profile of theexosome treatment. Given that exosomes encapsulate only 20% of siRNA,delivery with RVG-exosome appears to be more efficient than RVG-9Rdelivery as comparable mRNA knockdown and greater protein knockdown wasachieved with fivefold less siRNA without the corresponding level ofimmune stimulation. This experiment demonstrated the therapeuticpotential of RVG-exosome technology, which is potentially suited forlong-term silencing of genes related to neurodegenerative diseases. Theexosome delivery system of Alvarez-Erviti et al. may be applied todeliver the nucleic acid-targeting system of the present invention totherapeutic targets, especially neurodegenerative diseases. A dosage ofabout 100 to 1000 mg of nucleic acid-targeting system encapsulated inabout 100 to 1000 mg of RVG exosomes may be contemplated for the presentinvention.

El-Andaloussi et al. (Nature Protocols 7, 2112-2126 (2012)) discloseshow exosomes derived from cultured cells can be harnessed for deliveryof RNA in vitro and in vivo. This protocol first describes thegeneration of targeted exosomes through transfection of an expressionvector, comprising an exosomal protein fused with a peptide ligand.Next, El-Andaloussi et al. explain how to purify and characterizeexosomes from transfected cell supernatant. Next, El-Andaloussi et al.detail crucial steps for loading RNA into exosomes. Finally,El-Andaloussi et al. outline how to use exosomes to efficiently deliverRNA in vitro and in vivo in mouse brain. Examples of anticipated resultsin which exosome-mediated RNA delivery is evaluated by functional assaysand imaging are also provided. The entire protocol takes −3 weeks.Delivery or administration according to the invention may be performedusing exosomes produced from self-derived dendritic cells. From theherein teachings, this can be employed in the practice of the invention.

In another embodiment, the plasma exosomes of Wahlgren et al. (NucleicAcids Research, 2012, Vol. 40, No. 17 e130) are contemplated. Exosomesare nano-sized vesicles (30-90 nm in size) produced by many cell types,including dendritic cells (DC), B cells, T cells, mast cells, epithelialcells and tumor cells. These vesicles are formed by inward budding oflate endosomes and are then released to the extracellular environmentupon fusion with the plasma membrane. Because exosomes naturally carryRNA between cells, this property may be useful in gene therapy, and fromthis disclosure can be employed in the practice of the instantinvention.

Exosomes from plasma can be prepared by centrifugation of buffy coat at900 g for 20 min to isolate the plasma followed by harvesting cellsupernatants, centrifuging at 300 g for 10 min to eliminate cells and at16 500 g for 30 min followed by filtration through a 0.22 mm filter.Exosomes are pelleted by ultracentrifugation at 120 000 g for 70 min.Chemical transfection of siRNA into exosomes is carried out according tothe manufacturer's instructions in RNAi Human/Mouse Starter Kit(Quiagen, Hilden, Germany). siRNA is added to 100 ml PBS at a finalconcentration of 2 mmol/ml. After adding HiPerFect transfection reagent,the mixture is incubated for 10 min at RT. In order to remove the excessof micelles, the exosomes are re-isolated using aldehyde/sulfate latexbeads. The chemical transfection of nucleic acid-targeting system intoexosomes may be conducted similarly to siRNA. The exosomes may beco-cultured with monocytes and lymphocytes isolated from the peripheralblood of healthy donors. Therefore, it may be contemplated that exosomescontaining nucleic acid-targeting system may be introduced to monocytesand lymphocytes of and autologously reintroduced into a human.Accordingly, delivery or administration according to the invention maybe performed using plasma exosomes.

Liposomes

Delivery or administration according to the invention can be performedwith liposomes. Liposomes are spherical vesicle structures composed of auni- or multilamellar lipid bilayer surrounding internal aqueouscompartments and a relatively impermeable outer lipophilic phospholipidbilayer. Liposomes have gained considerable attention as drug deliverycarriers because they are biocompatible, nontoxic, can deliver bothhydrophilic and lipophilic drug molecules, protect their cargo fromdegradation by plasma enzymes, and transport their load acrossbiological membranes and the blood brain barrier (BBB) (see, e.g., Spuchand Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12pages, 2011. doi:10.1155/2011/469679 for review).

Liposomes can be made from several different types of lipids; however,phospholipids are most commonly used to generate liposomes as drugcarriers. Although liposome formation is spontaneous when a lipid filmis mixed with an aqueous solution, it can also be expedited by applyingforce in the form of shaking by using a homogenizer, sonicator, or anextrusion apparatus (see, e.g., Spuch and Navarro, Journal of DrugDelivery, vol. 2011, Article ID 469679, 12 pages, 2011.doi:10.1155/2011/469679 for review).

Several other additives may be added to liposomes in order to modifytheir structure and properties. For instance, either cholesterol orsphingomyelin may be added to the liposomal mixture in order to helpstabilize the liposomal structure and to prevent the leakage of theliposomal inner cargo. Further, liposomes are prepared from hydrogenatedegg phosphatidylcholine or egg phosphatidylcholine, cholesterol, anddicetyl phosphate, and their mean vesicle sizes were adjusted to about50 and 100 nm. (see, e.g., Spuch and Navarro, Journal of Drug Delivery,vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679for review).

A liposome formulation may be mainly comprised of natural phospholipidsand lipids such as 1,2-distearoyl-sn-glycero-3-phosphatidyl choline(DSPC), sphingomyelin, egg phosphatidylcholines andmonosialoganglioside. Since this formulation is made up of phospholipidsonly, liposomal formulations have encountered many challenges, one ofthe ones being the instability in plasma. Several attempts to overcomethese challenges have been made, specifically in the manipulation of thelipid membrane. One of these attempts focused on the manipulation ofcholesterol. Addition of cholesterol to conventional formulationsreduces rapid release of the encapsulated bioactive compound into theplasma or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) increasesthe stability (see, e.g., Spuch and Navarro, Journal of Drug Delivery,vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679for review).

In a particularly advantageous embodiment, Trojan Horse liposomes (alsoknown as Molecular Trojan Horses) are desirable and protocols may befound at cshprotocols.cshlp.org/content/2010/4/pdb.prot5407.long. Theseparticles allow delivery of a transgene to the entire brain after anintravascular injection. Without being bound by limitation, it isbelieved that neutral lipid particles with specific antibodiesconjugated to surface allow crossing of the blood brain barrier viaendocytosis. Applicant postulates utilizing Trojan Horse Liposomes todeliver the CRISPR family of nucleases to the brain via an intravascularinjection, which would allow whole brain transgenic animals without theneed for embryonic manipulation. About 1-5 g of DNA or RNA may becontemplated for in vivo administration in liposomes.

In another embodiment, the nucleic acid-targeting system or componentsthereof may be administered in liposomes, such as a stablenucleic-acid-lipid particle (SNALP) (see, e.g., Morrissey et al., NatureBiotechnology, Vol. 23, No. 8, August 2005). Daily intravenousinjections of about 1, 3 or 5 mg/kg/day of a specific nucleicacid-targeting system targeted in a SNALP are contemplated. The dailytreatment may be over about three days and then weekly for about fiveweeks. In another embodiment, a specific nucleic acid-targeting systemencapsulated SNALP) administered by intravenous injection to at doses ofabout 1 or 2.5 mg/kg are also contemplated (see, e.g., Zimmerman et al.,Nature Letters, Vol. 441, 4 May 2006). The SNALP formulation may containthe lipids 3-N-[(methoxypoly(ethylene glycol) 2000)carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-C-DMA),1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, in a2:40:10:48 molar percent ratio (see, e.g., Zimmerman et al., NatureLetters, Vol. 441, 4 May 2006).

In another embodiment, stable nucleic-acid-lipid particles (SNALPs) haveproven to be effective delivery molecules to highly vascularizedHepG2-derived liver tumors but not in poorly vascularized HCT-116derived liver tumors (see, e.g., Li, Gene Therapy (2012) 19, 775-780).The SNALP liposomes may be prepared by formulating D-Lin-DMA andPEG-C-DMA with distearoylphosphatidylcholine (DSPC), Cholesterol andsiRNA using a 25:1 lipid/siRNA ratio and a 48/40/10/2 molar ratio ofCholesterol/D-Lin-DMA/DSPC/PEG-C-DMA. The resulted SNALP liposomes areabout 80-100 nm in size.

In yet another embodiment, a SNALP may comprise synthetic cholesterol(Sigma-Aldrich, St Louis, Mo., USA), dipalmitoylphosphatidylcholine(Avanti Polar Lipids, Alabaster, Ala., USA), 3-N-[(w-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxypropylamine, andcationic 1,2-dilinoleyloxy-3-N,Ndimethylaminopropane (see, e.g.,Geisbert et al., Lancet 2010; 375: 1896-905). A dosage of about 2 mg/kgtotal nucleic acid-targeting system per dose administered as, forexample, a bolus intravenous infusion may be contemplated.

In yet another embodiment, a SNALP may comprise synthetic cholesterol(Sigma-Aldrich), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC;Avanti Polar Lipids Inc.), PEG-cDMA, and1,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMA) (see, e.g.,Judge, J. Clin. Invest. 119:661-673 (2009)). Formulations used for invivo studies may comprise a final lipid/RNA mass ratio of about 9:1.

The safety profile of RNAi nanomedicines has been reviewed by Barros andGollob of Alnylam Pharmaceuticals (see, e.g., Advanced Drug DeliveryReviews 64 (2012) 1730-1737). The stable nucleic acid lipid particle(SNALP) is comprised of four different lipids—an ionizable lipid(DLinDMA) that is cationic at low pH, a neutral helper lipid,cholesterol, and a diffusible polyethylene glycol (PEG)-lipid. Theparticle is approximately 80 nm in diameter and is charge-neutral atphysiologic pH. During formulation, the ionizable lipid serves tocondense lipid with the anionic RNA during particle formation. Whenpositively charged under increasingly acidic endosomal conditions, theionizable lipid also mediates the fusion of SNALP with the endosomalmembrane enabling release of RNA into the cytoplasm. The PEG-lipidstabilizes the particle and reduces aggregation during formulation, andsubsequently provides a neutral hydrophilic exterior that improvespharmacokinetic properties.

To date, two clinical programs have been initiated using SNALPformulations with RNA. Tekmira Pharmaceuticals recently completed aphase I single-dose study of SNALP-ApoB in adult volunteers withelevated LDL cholesterol. ApoB is predominantly expressed in the liverand jejunum and is essential for the assembly and secretion of VLDL andLDL. Seventeen subjects received a single dose of SNALP-ApoB (doseescalation across 7 dose levels). There was no evidence of livertoxicity (anticipated as the potential dose-limiting toxicity based onpreclinical studies). One (of two) subjects at the highest doseexperienced flu-like symptoms consistent with immune system stimulation,and the decision was made to conclude the trial.

Alnylam Pharmaceuticals has similarly advanced ALN-TTR01, which employsthe SNALP technology described above and targets hepatocyte productionof both mutant and wild-type TTR to treat TTR amyloidosis (ATTR). ThreeATTR syndromes have been described: familial amyloidotic polyneuropathy(FAP) and familial amyloidotic cardiomyopathy (FAC)—both caused byautosomal dominant mutations in TTR; and senile systemic amyloidosis(SSA) cause by wildtype TTR. A placebo-controlled, singledose-escalation phase I trial of ALN-TTR01 was recently completed inpatients with ATTR. ALN-TTR01 was administered as a 15-minute IVinfusion to 31 patients (23 with study drug and 8 with placebo) within adose range of 0.01 to 1.0 mg/kg (based on siRNA). Treatment was welltolerated with no significant increases in liver function tests.Infusion-related reactions were noted in 3 of 23 patients at ≥0.4 mg/kg;all responded to slowing of the infusion rate and all continued onstudy. Minimal and transient elevations of serum cytokines IL-6, IP-10and IL-1ra were noted in two patients at the highest dose of 1 mg/kg (asanticipated from preclinical and NHP studies). Lowering of serum TTR,the expected pharmacodynamics effect of ALN-TTR01, was observed at 1mg/kg.

In yet another embodiment, a SNALP may be made by solubilizing acationic lipid, DSPC, cholesterol and PEG-lipid e.g., in ethanol, e.g.,at a molar ratio of 40:10:40:10, respectively (see, Semple et al.,Nature Biotechnology, Volume 28 Number 2 Feb. 2010, pp. 172-177). Thelipid mixture was added to an aqueous buffer (50 mM citrate, pH 4) withmixing to a final ethanol and lipid concentration of 30% (vol/vol) and6.1 mg/ml, respectively, and allowed to equilibrate at 22° C. for 2 minbefore extrusion. The hydrated lipids were extruded through two stacked80 nm pore-sized filters (Nuclepore) at 22° C. using a Lipex Extruder(Northern Lipids) until a vesicle diameter of 70-90 nm, as determined bydynamic light scattering analysis, was obtained. This generally required1-3 passes. The siRNA (solubilized in a 50 mM citrate, pH 4 aqueoussolution containing 30% ethanol) was added to the pre-equilibrated (35°C.) vesicles at a rate of ˜5 ml/min with mixing. After a final targetsiRNA/lipid ratio of 0.06 (wt/wt) was reached, the mixture was incubatedfor a further 30 min at 35° C. to allow vesicle reorganization andencapsulation of the siRNA. The ethanol was then removed and theexternal buffer replaced with PBS (155 mM NaCl, 3 mM Na₂HPO₄, 1 mMKH₂PO₄, pH 7.5) by either dialysis or tangential flow diafiltration.siRNA were encapsulated in SNALP using a controlled step-wise dilutionmethod process. The lipid constituents of KC2-SNALP were DLin-KC2-DMA(cationic lipid), dipalmitoylphosphatidylcholine (DPPC; Avanti PolarLipids), synthetic cholesterol (Sigma) and PEG-C-DMA used at a molarratio of 57.1:7.1:34.3:1.4. Upon formation of the loaded particles,SNALP were dialyzed against PBS and filter sterilized through a 0.2 pmfilter before use. Mean particle sizes were 75-85 nm and 90-95% of thesiRNA was encapsulated within the lipid particles. The final siRNA/lipidratio in formulations used for in vivo testing was ˜0.15 (wt/wt).LNP-siRNA systems containing Factor VII siRNA were diluted to theappropriate concentrations in sterile PBS immediately before use and theformulations were administered intravenously through the lateral tailvein in a total volume of 10 ml/kg. This method and these deliverysystems may be extrapolated to the nucleic acid-targeting system of thepresent invention.

Other Lipids

Other cationic lipids, such as amino lipid2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) maybe utilized to encapsulate nucleic acid-targeting system or componentsthereof or nucleic acid molecule(s) coding therefor e.g., similar toSiRNA (see, e.g., Jayaraman, Angew. Chem. Int. Ed. 2012, 51, 8529-8533),and hence may be employed in the practice of the invention. A preformedvesicle with the following lipid composition may be contemplated: aminolipid, distearoylphosphatidylcholine (DSPC), cholesterol and(R)-2,3-bis(octadecyloxy) propyl-1-(methoxy poly(ethyleneglycol)2000)propylcarbamate (PEG-lipid) in the molar ratio 40/10/40/10,respectively, and a FVII siRNA/total lipid ratio of approximately 0.05(w/w). To ensure a narrow particle size distribution in the range of70-90 nm and a low polydispersity index of 0.11+0.04 (n=56), theparticles may be extruded up to three times through 80 nm membranesprior to adding the guide RNA. Particles containing the highly potentamino lipid 16 may be used, in which the molar ratio of the four lipidcomponents 16, DSPC, cholesterol and PEG-lipid (50/10/38.5/1.5) whichmay be further optimized to enhance in vivo activity.

Michael S D Kormann et al. (“Expression of therapeutic proteins afterdelivery of chemically modified mRNA in mice: Nature Biotechnology,Volume:29, Pages: 154-157 (2011)) describes the use of lipid envelopesto deliver RNA. Use of lipid envelopes is also preferred in the presentinvention.

In another embodiment, lipids may be formulated with the nucleicacid-targeting system of the present invention or component(s) thereofor nucleic acid molecule(s) coding therefor to form lipid nanoparticles(LNPs). Lipids include, but are not limited to, DLin-KC2-DMA4, C12-200and colipids disteroylphosphatidyl choline, cholesterol, and PEG-DMG maybe formulated with RNA-targeting system instead of siRNA (see, e.g.,Novobrantseva, Molecular Therapy-Nucleic Acids (2012) 1, e4;doi:10.1038/mtna.2011.3) using a spontaneous vesicle formationprocedure. The component molar ratio may be about 50/10/38.5/1.5(DLin-KC2-DMA or C12-200/disteroylphosphatidylcholine/cholesterol/PEG-DMG). The final lipid:siRNA weight ratio may be˜12:1 and 9:1 in the case of DLin-KC2-DMA and C12-200 lipid particles(LNPs), respectively. The formulations may have mean particle diametersof ˜80 nm with >90% entrapment efficiency. A 3 mg/kg dose may becontemplated.

Tekmira has a portfolio of approximately 95 patent families, in the U.S.and abroad, that are directed to various aspects of LNPs and LNPformulations (see, e.g., U.S. Pat. Nos. 7,982,027; 7,799,565; 8,058,069;8,283,333; 7,901,708; 7,745,651; 7,803,397; 8,101,741; 8,188,263;7,915,399; 8,236,943 and 7,838,658 and European Pat. Nos 1766035;1519714; 1781593 and 1664316), all of which may be used and/or adaptedto the present invention.

The nucleic acid-targeting system or components thereof or nucleic acidmolecule(s) coding therefor may be delivered encapsulated in PLGAMicrospheres such as that further described in US published applications20130252281 and 20130245107 and 20130244279 (assigned to ModernaTherapeutics) which relate to aspects of formulation of compositionscomprising modified nucleic acid molecules which may encode a protein, aprotein precursor, or a partially or fully processed form of the proteinor a protein precursor. The formulation may have a molar ratio50:10:38.5:1.5-3.0 (cationic lipid:fusogenic lipid:cholesterol:PEGlipid). The PEG lipid may be selected from, but is not limited toPEG-c-DOMG, PEG-DMG. The fusogenic lipid may be DSPC. See also, Schrumet al., Delivery and Formulation of Engineered Nucleic Acids, USpublished application 20120251618.

Nanomerics' technology addresses bioavailability challenges for a broadrange of therapeutics, including low molecular weight hydrophobic drugs,peptides, and nucleic acid based therapeutics (plasmid, siRNA, miRNA).Specific administration routes for which the technology has demonstratedclear advantages include the oral route, transport across theblood-brain-barrier, delivery to solid tumours, as well as to the eye.See, e.g., Mazza et al., 2013, ACS Nano. 2013 Feb. 26; 7(2):1016-26;Uchegbu and Siew, 2013, J Pharm Sci. 102(2):305-10 and Lalatsa et al.,2012, J Control Release. 2012 Jul. 20; 161(2):523-36.

US Patent Publication No. 20050019923 describes cationic dendrimers fordelivering bioactive molecules, such as polynucleotide molecules,peptides and polypeptides and/or pharmaceutical agents, to a mammalianbody. The dendrimers are suitable for targeting the delivery of thebioactive molecules to, for example, the liver, spleen, lung, kidney orheart (or even the brain). Dendrimers are synthetic 3-dimensionalmacromolecules that are prepared in a step-wise fashion from simplebranched monomer units, the nature and functionality of which can beeasily controlled and varied. Dendrimers are synthesized from therepeated addition of building blocks to a multifunctional core(divergent approach to synthesis), or towards a multifunctional core(convergent approach to synthesis) and each addition of a 3-dimensionalshell of building blocks leads to the formation of a higher generationof the dendrimers. Polypropylenimine dendrimers start from adiaminobutane core to which is added twice the number of amino groups bya double Michael addition of acrylonitrile to the primary aminesfollowed by the hydrogenation of the nitriles. This results in adoubling of the amino groups. Polypropylenimine dendrimers contain 100%protonable nitrogens and up to 64 terminal amino groups (generation 5,DAB 64). Protonable groups are usually amine groups which are able toaccept protons at neutral pH. The use of dendrimers as gene deliveryagents has largely focused on the use of the polyamidoamine andphosphorous containing compounds with a mixture of amine/amide orN—P(O₂)S as the conjugating units respectively with no work beingreported on the use of the lower generation polypropylenimine dendrimersfor gene delivery. Polypropylenimine dendrimers have also been studiedas pH sensitive controlled release systems for drug delivery and fortheir encapsulation of guest molecules when chemically modified byperipheral amino acid groups. The cytotoxicity and interaction ofpolypropylenimine dendrimers with DNA as well as the transfectionefficacy of DAB 64 has also been studied.

US Patent Publication No. 20050019923 is based upon the observationthat, contrary to earlier reports, cationic dendrimers, such aspolypropylenimine dendrimers, display suitable properties, such asspecific targeting and low toxicity, for use in the targeted delivery ofbioactive molecules, such as genetic material. In addition, derivativesof the cationic dendrimer also display suitable properties for thetargeted delivery of bioactive molecules. See also, Bioactive Polymers,US published application 20080267903, which discloses “Various polymers,including cationic polyamine polymers and dendrimeric polymers, areshown to possess anti-proliferative activity, and may therefore beuseful for treatment of disorders characterised by undesirable cellularproliferation such as neoplasms and tumours, inflammatory disorders(including autoimmune disorders), psoriasis and atherosclerosis. Thepolymers may be used alone as active agents, or as delivery vehicles forother therapeutic agents, such as drug molecules or nucleic acids forgene therapy. In such cases, the polymers' own intrinsic anti-tumouractivity may complement the activity of the agent to be delivered.” Thedisclosures of these patent publications may be employed in conjunctionwith herein teachings for delivery of nucleic acid-targetingsystem(s) orcomponent(s) thereof or nucleic acid molecule(s) coding therefor.

Supercharged Proteins

Supercharged proteins are a class of engineered or naturally occurringproteins with unusually high positive or negative net theoretical chargeand may be employed in delivery of nucleic acid-targeting system(s) orcomponent(s) thereof or nucleic acid molecule(s) coding therefor. Bothsupernegatively and superpositively charged proteins exhibit aremarkable ability to withstand thermally or chemically inducedaggregation. Superpositively charged proteins are also able to penetratemammalian cells. Associating cargo with these proteins, such as plasmidDNA, RNA, or other proteins, can enable the functional delivery of thesemacromolecules into mammalian cells both in vitro and in vivo. DavidLiu's lab reported the creation and characterization of superchargedproteins in 2007 (Lawrence et al., 2007, Journal of the AmericanChemical Society 129, 10110-10112).

The nonviral delivery of RNA and plasmid DNA into mammalian cells arevaluable both for research and therapeutic applications (Akinc et al.,2010, Nat. Biotech. 26, 561-569). Purified+36 GFP protein (or othersuperpositively charged protein) is mixed with RNAs in the appropriateserum-free media and allowed to complex prior addition to cells.Inclusion of serum at this stage inhibits formation of the superchargedprotein-RNA complexes and reduces the effectiveness of the treatment.The following protocol has been found to be effective for a variety ofcell lines (McNaughton et al., 2009, Proc. Natl. Acad. Sci. USA 106,6111-6116). However, pilot experiments varying the dose of protein andRNA should be performed to optimize the procedure for specific celllines.

(1) One day before treatment, plate 1×10⁵ cells per well in a 48-wellplate.

(2) On the day of treatment, dilute purified +36 GFP protein inserumfree media to a final concentration 200 nM. Add RNA to a finalconcentration of 50 nM. Vortex to mix and incubate at room temperaturefor 10 min.

(3) During incubation, aspirate media from cells and wash once with PBS.

(4) Following incubation of +36 GFP and RNA, add the protein-RNAcomplexes to cells.

(5) Incubate cells with complexes at 37° C. for 4 h.

(6) Following incubation, aspirate the media and wash three times with20 U/mL heparin PBS. Incubate cells with serum-containing media for afurther 48 h or longer depending upon the assay for activity.

(7) Analyze cells by immunoblot, qPCR, phenotypic assay, or otherappropriate method.

David Liu's lab has further found +36 GFP to be an effective plasmiddelivery reagent in a range of cells. As plasmid DNA is a larger cargothan siRNA, proportionately more +36 GFP protein is required toeffectively complex plasmids. For effective plasmid delivery Applicantshave developed a variant of +36 GFP bearing a C-terminal HA2 peptidetag, a known endosome-disrupting peptide derived from the influenzavirus hemagglutinin protein. The following protocol has been effectivein a variety of cells, but as above it is advised that plasmid DNA andsupercharged protein doses be optimized for specific cell lines anddelivery applications.

(1) One day before treatment, plate 1×10⁵ per well in a 48-well plate.

(2) On the day of treatment, dilute purified

36 GFP protein in serumfree media to a final concentration 2 mM. Add 1mg of plasmid DNA. Vortex to mix and incubate at room temperature for 10min.

(3) During incubation, aspirate media from cells and wash once with PBS.

(4) Following incubation of

36 GFP and plasmid DNA, gently add the protein-DNA complexes to cells.

(5) Incubate cells with complexes at 37 C for 4 h.

(6) Following incubation, aspirate the media and wash with PBS. Incubatecells in serum-containing media and incubate for a further 24-48 h.

(7) Analyze plasmid delivery (e.g., by plasmid-driven gene expression)as appropriate.

See also, e.g., McNaughton et al., Proc. Natl. Acad. Sci. USA 106,6111-6116 (2009); Cronican et al., ACS Chemical Biology 5, 747-752(2010); Cronican et al., Chemistry & Biology 18, 833-838 (2011);Thompson et al., Methods in Enzymology 503, 293-319 (2012); Thompson, D.B., et al., Chemistry & Biology 19 (7), 831-843 (2012). The methods ofthe super charged proteins may be used and/or adapted for delivery ofthe nucleic acid-targeting system of the present invention. Thesesystems of Dr. Lui and documents herein in conjunction with hereinteachings can be employed in the delivery of nucleic acid-targetingsystem(s) or component(s) thereof or nucleic acid molecule(s) codingtherefor.

Cell Penetrating Peptides (CPPs)

In yet another embodiment, cell penetrating peptides (CPPs) arecontemplated for the delivery of the CRISPR Cas system. CPPs are shortpeptides that facilitate cellular uptake of various molecular cargo(from nanosize particles to small chemical molecules and large fragmentsof DNA). The term “cargo” as used herein includes but is not limited tothe group consisting of therapeutic agents, diagnostic probes, peptides,nucleic acids, antisense oligonucleotides, plasmids, proteins, particlesincluding nanoparticles, liposomes, chromophores, small molecules andradioactive materials. In aspects of the invention, the cargo may alsocomprise any component of the CRISPR Cas system or the entire functionalCRISPR Cas system. Aspects of the present invention further providemethods for delivering a desired cargo into a subject comprising: (a)preparing a complex comprising the cell penetrating peptide of thepresent invention and a desired cargo, and (b) orally, intraarticularly,intraperitoneally, intrathecally, intraarterially, intranasally,intraparenchymally, subcutaneously, intramuscularly, intravenously,dermally, intrarectally, or topically administering the complex to asubject. The cargo is associated with the peptides either throughchemical linkage via covalent bonds or through non-covalentinteractions.

The function of the CPPs are to deliver the cargo into cells, a processthat commonly occurs through endocytosis with the cargo delivered to theendosomes of living mammalian cells. Cell-penetrating peptides are ofdifferent sizes, amino acid sequences, and charges but all CPPs have onedistinct characteristic, which is the ability to translocate the plasmamembrane and facilitate the delivery of various molecular cargoes to thecytoplasm or an organelle. CPP translocation may be classified intothree main entry mechanisms: direct penetration in the membrane,endocytosis-mediated entry, and translocation through the formation of atransitory structure. CPPs have found numerous applications in medicineas drug delivery agents in the treatment of different diseases includingcancer and virus inhibitors, as well as contrast agents for celllabeling. Examples of the latter include acting as a carrier for GFP,MRI contrast agents, or quantum dots. CPPs hold great potential as invitro and in vivo delivery vectors for use in research and medicine.CPPs typically have an amino acid composition that either contains ahigh relative abundance of positively charged amino acids such as lysineor arginine or has sequences that contain an alternating pattern ofpolar/charged amino acids and non-polar, hydrophobic amino acids. Thesetwo types of structures are referred to as polycationic or amphipathic,respectively. A third class of CPPs are the hydrophobic peptides,containing only a polar residues, with low net charge or havehydrophobic amino acid groups that are crucial for cellular uptake. Oneof the initial CPPs discovered was the trans-activating transcriptionalactivator (Tat) from Human Immunodeficiency Virus 1 (HIV-1) which wasfound to be efficiently taken up from the surrounding media by numerouscell types in culture. Since then, the number of known CPPs has expandedconsiderably and small molecule synthetic analogues with more effectiveprotein transduction properties have been generated. CPPs include butare not limited to Penetratin, Tat (48-60), Transportan, and (R-AhX-R)4)(SEQ ID NO: 30) (Ahx=aminohexanoyl).

U.S. Pat. No. 8,372,951, provides a CPP derived from eosinophil cationicprotein (ECP) which exhibits highly cell-penetrating efficiency and lowtoxicity. Aspects of delivering the CPP with its cargo into a vertebratesubject are also provided. Further aspects of CPPs and their deliveryare described in U.S. Pat. Nos. 8,575,305; 8,614,194 and 8,044,019. CPPscan be used to deliver the CRISPR-Cas system or components thereof. ThatCPPs can be employed to deliver the CRISPR-Cas system or componentsthereof is also provided in the manuscript “Gene disruption bycell-penetrating peptide-mediated delivery of Cas9 protein and guideRNA”, by Suresh Ramakrishna, Abu-Bonsrah Kwaku Dad, Jagadish Beloor, etal. Genome Res. 2014 Apr. 2. [Epub ahead of print], incorporated byreference in its entirety, wherein it is demonstrated that treatmentwith CPP-conjugated recombinant Cas9 protein and CPP-complexed guideRNAs lead to endogenous gene disruptions in human cell lines. In thepaper the Cas9 protein was conjugated to CPP via a thioether bond,whereas the guide RNA was complexed with CPP, forming condensed,positively charged particles. It was shown that simultaneous andsequential treatment of human cells, including embryonic stem cells,dermal fibroblasts, HEK293T cells, HeLa cells, and embryonic carcinomacells, with the modified Cas9 and guide RNA led to efficient genedisruptions with reduced off-target mutations relative to plasmidtransfections.

Implantable Devices

In another embodiment, implantable devices are also contemplated fordelivery of the nucleic acid-targeting system or component(s) thereof ornucleic acid molecule(s) coding therefor. For example, US PatentPublication 20110195123 discloses an implantable medical device whichelutes a drug locally and in prolonged period is provided, includingseveral types of such a device, the treatment modes of implementationand methods of implantation. The device comprising of polymericsubstrate, such as a matrix for example, that is used as the devicebody, and drugs, and in some cases additional scaffolding materials,such as metals or additional polymers, and materials to enhancevisibility and imaging. An implantable delivery device can beadvantageous in providing release locally and over a prolonged period,where drug is released directly to the extracellular matrix (ECM) of thediseased area such as tumor, inflammation, degeneration or forsymptomatic objectives, or to injured smooth muscle cells, or forprevention. One kind of drug is RNA, as disclosed above, and this systemmay be used and/or adapted to the nucleic acid-targeting system of thepresent invention. The modes of implantation in some embodiments areexisting implantation procedures that are developed and used today forother treatments, including brachytherapy and needle biopsy. In suchcases the dimensions of the new implant described in this invention aresimilar to the original implant. Typically a few devices are implantedduring the same treatment procedure.

US Patent Publication 20110195123, provides a drug delivery implantableor insertable system, including systems applicable to a cavity such asthe abdominal cavity and/or any other type of administration in whichthe drug delivery system is not anchored or attached, comprising abiostable and/or degradable and/or bioabsorbable polymeric substrate,which may for example optionally be a matrix. It should be noted thatthe term “insertion” also includes implantation. The drug deliverysystem is preferably implemented as a “Loder” as described in US PatentPublication 20110195123.

The polymer or plurality of polymers are biocompatible, incorporating anagent and/or plurality of agents, enabling the release of agent at acontrolled rate, wherein the total volume of the polymeric substrate,such as a matrix for example, in some embodiments is optionally andpreferably no greater than a maximum volume that permits a therapeuticlevel of the agent to be reached. As a non-limiting example, such avolume is preferably within the range of 0.1 m³ to 1000 mm³, as requiredby the volume for the agent load. The Loder may optionally be larger,for example when incorporated with a device whose size is determined byfunctionality, for example and without limitation, a knee joint, anintra-uterine or cervical ring and the like.

The drug delivery system (for delivering the composition) is designed insome embodiments to preferably employ degradable polymers, wherein themain release mechanism is bulk erosion; or in some embodiments, nondegradable, or slowly degraded polymers are used, wherein the mainrelease mechanism is diffusion rather than bulk erosion, so that theouter part functions as membrane, and its internal part functions as adrug reservoir, which practically is not affected by the surroundingsfor an extended period (for example from about a week to about a fewmonths). Combinations of different polymers with different releasemechanisms may also optionally be used. The concentration gradient atthe surface is preferably maintained effectively constant during asignificant period of the total drug releasing period, and therefore thediffusion rate is effectively constant (termed “zero mode” diffusion).By the term “constant” it is meant a diffusion rate that is preferablymaintained above the lower threshold of therapeutic effectiveness, butwhich may still optionally feature an initial burst and/or mayfluctuate, for example increasing and decreasing to a certain degree.The diffusion rate is preferably so maintained for a prolonged period,and it can be considered constant to a certain level to optimize thetherapeutically effective period, for example the effective silencingperiod.

The drug delivery system optionally and preferably is designed to shieldthe nucleotide based therapeutic agent from degradation, whetherchemical in nature or due to attack from enzymes and other factors inthe body of the subject.

The drug delivery system of US Patent Publication 20110195123 isoptionally associated with sensing and/or activation appliances that areoperated at and/or after implantation of the device, by non and/orminimally invasive methods of activation and/oracceleration/deceleration, for example optionally including but notlimited to thermal heating and cooling, laser beams, and ultrasonic,including focused ultrasound and/or RF (radiofrequency) methods ordevices.

According to some embodiments of US Patent Publication 20110195123, thesite for local delivery may optionally include target sitescharacterized by high abnormal proliferation of cells, and suppressedapoptosis, including tumors, active and/or chronic inflammation andinfection including autoimmune diseases states, degenerating tissueincluding muscle and nervous tissue, chronic pain, degenerative sites,and location of bone fractures and other wound locations for enhancementof regeneration of tissue, and injured cardiac, smooth and striatedmuscle.

The site for implantation of the composition, or target site, preferablyfeatures a radius, area and/or volume that is sufficiently small fortargeted local delivery. For example, the target site optionally has adiameter in a range of from about 0.1 mm to about 5 cm.

The location of the target site is preferably selected for maximumtherapeutic efficacy. For example, the composition of the drug deliverysystem (optionally with a device for implantation as described above) isoptionally and preferably implanted within or in the proximity of atumor environment, or the blood supply associated thereof.

For example the composition (optionally with the device) is optionallyimplanted within or in the proximity to pancreas, prostate, breast,liver, via the nipple, within the vascular system and so forth.

The target location is optionally selected from the group comprising,consisting essentially of, or consisting of (as non-limiting examplesonly, as optionally any site within the body may be suitable forimplanting a Loder): 1. brain at degenerative sites like in Parkinson orAlzheimer disease at the basal ganglia, white and gray matter; 2. spineas in the case of amyotrophic lateral sclerosis (ALS); 3. uterine cervixto prevent HPV infection; 4. active and chronic inflammatory joints; 5.dermis as in the case of psoriasis; 6. sympathetic and sensoric nervoussites for analgesic effect; 7. Intra osseous implantation; 8. acute andchronic infection sites; 9. Intra vaginal; 10. Inner ear-auditorysystem, labyrinth of the inner ear, vestibular system; 11. Intratracheal; 12. Intra-cardiac; coronary, epicardiac; 13. urinary bladder;14. biliary system; 15. parenchymal tissue including and not limited tothe kidney, liver, spleen; 16. lymph nodes; 17. salivary glands; 18.dental gums; 19. Intra-articular (into joints); 20. Intra-ocular; 21.Brain tissue; 22. Brain ventricles; 23. Cavities, including abdominalcavity (for example but without limitation, for ovary cancer); 24. Intraesophageal and 25. Intra rectal.

Optionally insertion of the system (for example a device containing thecomposition) is associated with injection of material to the ECM at thetarget site and the vicinity of that site to affect local pH and/ortemperature and/or other biological factors affecting the diffusion ofthe drug and/or drug kinetics in the ECM, of the target site and thevicinity of such a site.

Optionally, according to some embodiments, the release of said agentcould be associated with sensing and/or activation appliances that areoperated prior and/or at and/or after insertion, by non and/or minimallyinvasive and/or else methods of activation and/oracceleration/deceleration, including laser beam, radiation, thermalheating and cooling, and ultrasonic, including focused ultrasound and/orRF (radiofrequency) methods or devices, and chemical activators.

According to other embodiments of US Patent Publication 20110195123, thedrug preferably comprises a RNA, for example for localized cancer casesin breast, pancreas, brain, kidney, bladder, lung, and prostate asdescribed below. Although exemplified with RNAi, many drugs areapplicable to be encapsulated in Loder, and can be used in associationwith this invention, as long as such drugs can be encapsulated with theLoder substrate, such as a matrix for example, and this system may beused and/or adapted to deliver the nucleic acid-targeting system of thepresent invention.

As another example of a specific application, neuro and musculardegenerative diseases develop due to abnormal gene expression. Localdelivery of RNAs may have therapeutic properties for interfering withsuch abnormal gene expression. Local delivery of anti apoptotic, antiinflammatory and anti degenerative drugs including small drugs andmacromolecules may also optionally be therapeutic. In such cases theLoder is applied for prolonged release at constant rate and/or through adedicated device that is implanted separately. All of this may be usedand/or adapted to the nucleic acid-targeting system of the presentinvention.

As yet another example of a specific application, psychiatric andcognitive disorders are treated with gene modifiers. Gene knockdown is atreatment option. Loders locally delivering agents to central nervoussystem sites are therapeutic options for psychiatric and cognitivedisorders including but not limited to psychosis, bi-polar diseases,neurotic disorders and behavioral maladies. The Loders could alsodeliver locally drugs including small drugs and macromolecules uponimplantation at specific brain sites. All of this may be used and/oradapted to the nucleic acid-targeting system of the present invention.

As another example of a specific application, silencing of innate and/oradaptive immune mediators at local sites enables the prevention of organtransplant rejection. Local delivery of RNAs and immunomodulatingreagents with the Loder implanted into the transplanted organ and/or theimplanted site renders local immune suppression by repelling immunecells such as CD8 activated against the transplanted organ. All of thismay be used and/or adapted to the nucleic acid-targeting system of thepresent invention.

As another example of a specific application, vascular growth factorsincluding VEGFs and angiogenin and others are essential forneovascularization. Local delivery of the factors, peptides,peptidomimetics, or suppressing their repressors is an importanttherapeutic modality; silencing the repressors and local delivery of thefactors, peptides, macromolecules and small drugs stimulatingangiogenesis with the Loder is therapeutic for peripheral, systemic andcardiac vascular disease.

The method of insertion, such as implantation, may optionally already beused for other types of tissue implantation and/or for insertions and/orfor sampling tissues, optionally without modifications, or alternativelyoptionally only with non-major modifications in such methods. Suchmethods optionally include but are not limited to brachytherapy methods,biopsy, endoscopy with and/or without ultrasound, such as ERCP,stereotactic methods into the brain tissue, Laparoscopy, includingimplantation with a laparoscope into joints, abdominal organs, thebladder wall and body cavities.

Implantable device technology herein discussed can be employed withherein teachings and hence by this disclosure and the knowledge in theart, CRISPR-Cas system or components thereof or nucleic acid moleculesthereof or encoding or providing components may be delivered via animplantable device.

Patient-Specific Screening Methods

A nucleic acid-targeting system that targets RNA, e.g., trinucleotiderepeats can be used to screen patients or patent samples for thepresence of such repeats. The repeats can be the target of the RNA ofthe nucleic acid-targeting system, and if there is binding thereto bythe nucleic acid-targeting system, that binding can be detected, tothereby indicate that such a repeat is present. Thus, a nucleicacid-targeting system can be used to screen patients or patient samplesfor the presence of the repeat. The patient can then be administeredsuitable compound(s) to address the condition; or, can be administered anucleic acid-targeting system to bind to and cause insertion, deletionor mutation and alleviate the condition.

The invention uses nucleic acids to bind target RNA sequences.

CRISPR Effector Protein mRNA and Guide RNA

CRISPR effector protein mRNA and guide RNA might also be deliveredseparately. CRISPR effector protein mRNA can be delivered prior to theguide RNA to give time for CRISPR effector protein to be expressed.CRISPR effector protein mRNA might be administered 1-12 hours(preferably around 2-6 hours) prior to the administration of guide RNA.

Alternatively, CRISPR effector protein mRNA and guide RNA can beadministered together. Advantageously, a second booster dose of guideRNA can be administered 1-12 hours (preferably around 2-6 hours) afterthe initial administration of CRISPR effector protein mRNA +guide RNA.

The CRISPR effector protein of the present invention, i.e. a C2c1 orC2c3 effector protein is sometimes referred to herein as a CRISPREnzyme. It will be appreciated that the effector protein is based on orderived from an enzyme, so the term ‘effector protein’ certainlyincludes ‘enzyme’ in some embodiments. However, it will also beappreciated that the effector protein may, as required in someembodiments, have DNA or RNA binding, but not necessarily cutting ornicking, activity, including a dead-Cas effector protein function.

Additional administrations of CRISPR effector protein mRNA and/or guideRNA might be useful to achieve the most efficient levels of genomemodification. In some embodiments, phenotypic alteration is preferablythe result of genome modification when a genetic disease is targeted,especially in methods of therapy and preferably where a repair templateis provided to correct or alter the phenotype.

In some embodiments diseases that may be targeted include thoseconcerned with disease-causing splice defects.

In some embodiments, cellular targets include HemopoieticStem/Progenitor Cells (CD34+); Human T cells; and Eye (retinalcells)—for example photoreceptor precursor cells.

In some embodiments Gene targets include: Human Beta Globin—HBB (fortreating Sickle Cell Anemia, including by stimulating gene-conversion(using closely related HBD gene as an endogenous template)); CD3(T-Cells); and CEP920-retina (eye).

In some embodiments disease targets also include: cancer; Sickle CellAnemia (based on a point mutation); HIV; Beta-Thalassemia; andophthalmic or ocular disease—for example Leber Congenital Amaurosis(LCA)-causing Splice Defect.

In some embodiments delivery methods include: Cationic Lipid Mediated“direct” delivery of Enzyme-Guide complex (RiboNucleoProtein) andelectroporation of plasmid DNA.

Inventive methods can further comprise delivery of templates, such asrepair templates, which may be dsODN or ssODN, see below. Delivery oftemplates may be via the cotemporaneous or separate from delivery of anyor all the CRISPR effector protein or guide and via the same deliverymechanism or different. In some embodiments, it is preferred that thetemplate is delivered together with the guide, and, preferably, also theCRISPR effector protein. An example may be an AAV vector.

Inventive methods can further comprise: (a) delivering to the cell adouble-stranded oligodeoxynucleotide (dsODN) comprising overhangscomplimentary to the overhangs created by said double strand break,wherein said dsODN is integrated into the locus of interest; or—(b)delivering to the cell a single-stranded oligodeoxynucleotide (ssODN),wherein said ssODN acts as a template for homology directed repair ofsaid double strand break. Inventive methods can be for the prevention ortreatment of disease in an individual, optionally wherein said diseaseis caused by a defect in said locus of interest. Inventive methods canbe conducted in vivo in the individual or ex vivo on a cell taken fromthe individual, optionally wherein said cell is returned to theindividual.

For minimization of toxicity and off-target effect, it will be importantto control the concentration of CRISPR effector protein mRNA and guideRNA delivered. Optimal concentrations of CRISPR effector protein mRNAand guide RNA can be determined by testing different concentrations in acellular or animal model and using deep sequencing the analyze theextent of modification at potential off-target genomic loci. Forexample, for the guide sequence targeting 5′-GAGTCCGAGCAGAAGAAGAA-3′(SEQ ID NO: 31) in the EMX1 gene of the human genome, deep sequencingcan be used to assess the level of modification at the following twooff-target loci, 1: 5′-GAGTCCTAGCAGGAGAAGAA-3′ (SEQ ID NO: 32) and 2:5′-GAGTCTAAGCAGAAGAAGAA-3′ (SEQ ID NO: 33). The concentration that givesthe highest level of on-target modification while minimizing the levelof off-target modification should be chosen for in vivo delivery.

Inducible Systems

In some embodiments, a CRISPR effector protein may form a component ofan inducible system. The inducible nature of the system would allow forspatiotemporal control of gene editing or gene expression using a formof energy. The form of energy may include but is not limited toelectromagnetic radiation, sound energy, chemical energy and thermalenergy. Examples of inducible system include tetracycline induciblepromoters (Tet-On or Tet-Off), small molecule two-hybrid transcriptionactivations systems (FKBP, ABA, etc), or light inducible systems(Phytochrome, LOV domains, or cryptochrome). In one embodiment, theCRISPR effector protein may be a part of a Light InducibleTranscriptional Effector (LITE) to direct changes in transcriptionalactivity in a sequence-specific manner. The components of a light mayinclude a CRISPR effector protein, a light-responsive cytochromeheterodimer (e.g. from Arabidopsis thaliana), and a transcriptionalactivation/repression domain. Further examples of inducible DNA bindingproteins and methods for their use are provided in U.S. 61/736,465 andU.S. 61/721,283, and WO 2014018423 A2 which is hereby incorporated byreference in its entirety.

Self-Inactivating Systems

Once all copies of a gene in the genome of a cell have been edited,continued CRISPR/C2c1 or C2c3 effector protein expression in that cellis no longer necessary. Indeed, sustained expression would beundesirable in case of off-target effects at unintended genomic sites,etc. Thus time-limited expression would be useful. Inducible expressionoffers one approach, but in addition Applicants have engineered aSelf-Inactivating CRISPR system that relies on the use of a non-codingguide target sequence within the CRISPR vector itself. Thus, afterexpression begins, the CRISPR system will lead to its own destruction,but before destruction is complete it will have time to edit the genomiccopies of the target gene (which, with a normal point mutation in adiploid cell, requires at most two edits). Simply, the self inactivatingCRISPR-Cas system includes additional RNA (i.e., guide RNA) that targetsthe coding sequence for the CRISPR effector protein itself or thattargets one or more non-coding guide target sequences complementary tounique sequences present in one or more of the following:

(a) within the promoter driving expression of the non-coding RNAelements,

(b) within the promoter driving expression of the C2c1 or C2c3 effectorprotein gene,

(c) within 100 bp of the ATG translational start codon in the C2c1 orC2c3 effector protein coding sequence,

(d) within the inverted terminal repeat (iTR) of a viral deliveryvector, e.g., in the AAV genome.

Furthermore, that RNA can be delivered via a vector, e.g., a separatevector or the same vector that is encoding the CRISPR complex. Whenprovided by a separate vector, the CRISPR RNA that targets Casexpression can be administered sequentially or simultaneously. Whenadministered sequentially, the CRISPR RNA that targets Cas expression isto be delivered after the CRISPR RNA that is intended for e.g. geneediting or gene engineering. This period may be a period of minutes(e.g. 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 60minutes). This period may be a period of hours (e.g. 2 hours, 4 hours, 6hours, 8 hours, 12 hours, 24 hours). This period may be a period of days(e.g. 2 days, 3 days, 4 days, 7 days). This period may be a period ofweeks (e.g. 2 weeks, 3 weeks, 4 weeks). This period may be a period ofmonths (e.g. 2 months, 4 months, 8 months, 12 months). This period maybe a period of years (2 years, 3 years, 4 years). Where the guide RNAtargets the sequences encoding expression of the Cas protein, theeffector protein becomes impeded and the system becomes selfinactivating. In the same manner, CRISPR RNA that targets Cas expressionapplied via, for example liposome, lipofection, particles, microvesiclesas explained herein, may be administered sequentially or simultaneously.Similarly, self-inactivation may be used for inactivation of one or moreguide RNA used to target one or more targets.

In some aspects, a single gRNA is provided that is capable ofhybridization to a sequence downstream of a CRISPR effector proteinstart codon, whereby after a period of time there is a loss of theCRISPR effector protein expression. In some aspects, one or more gRNA(s)are provided that are capable of hybridization to one or more coding ornon-coding regions of the polynucleotide encoding the CRISPR-Cas system,whereby after a period of time there is a inactivation of one or more,or in some cases all, of the CRISPR-Cas system. In some aspects of thesystem, and not to be limited by theory, the cell may comprise aplurality of CRISPR-Cas complexes, wherein a first subset of CRISPRcomplexes comprise a first guide RNA capable of targeting a genomiclocus or loci to be edited, and a second subset of CRISPR complexescomprise at least one second guide RNA capable of targeting thepolynucleotide encoding the CRISPR-Cas system, wherein the first subsetof CRISPR-Cas complexes mediate editing of the targeted genomic locus orloci and the second subset of CRISPR complexes eventually inactivate theCRISPR-Cas system, thereby inactivating further CRISPR-Cas expression inthe cell.

Thus the invention provides a CRISPR-Cas system comprising one or morevectors for delivery to a eukaryotic cell, wherein the vector(s)encode(s): (i) a CRISPR effector protein; (ii) a first guide RNA capableof hybridizing to a target sequence in the cell; (iii) a second guideRNA capable of hybridizing to one or more target sequence(s) in thevector which encodes the CRISPR effector protein; thus differing only bythe guide sequence, wherein, when expressed within the cell: the firstguide RNA directs sequence-specific binding of a first CRISPR complex tothe target sequence in the cell; the second guide RNA directssequence-specific binding of a second CRISPR complex to the targetsequence in the vector which encodes the CRISPR effector protein; theCRISPR complexes comprise a CRISPR effector protein bound to a guideRNA, such that a guide RNA can hybridize to its target sequence; and thesecond CRISPR complex inactivates the CRISPR-Cas system to preventcontinued expression of the CRISPR effector protein by the cell.

Further characteristics of the vector(s), the encoded enzyme, the guidesequences, etc. are disclosed elsewhere herein. The system can encode(i) a CRISPR enzyme, more particularly C2c1 or C2c3; (ii) a first gRNAcomprising a sequence capable of hybridizing to a first target sequencein the cell, (iii) a second guide RNA capable of hybridizing to thevector which encodes the CRISPR enzyme. Similarly, the enzyme caninclude one or more NLS, etc.

The various coding sequences (CRISPR effector protein and guide RNAs)can be included on a single vector or on multiple vectors. For instance,it is possible to encode the effector protein on one vector and thevarious RNA sequences on another vector, or to encode the effectorprotein and one guide RNA on one vector, and the remaining guide RNA onanother vector, or any other permutation. In general, a system using atotal of one or two different vectors is preferred.

Where multiple vectors are used, it is possible to deliver them inunequal numbers, and ideally with an excess of a vector which encodesthe first guide RNA relative to the second guide RNA, thereby assistingin delaying final inactivation of the CRISPR system until genome editinghas had a chance to occur.

The first guide RNA can target any target sequence of interest within agenome, as described elsewhere herein. The second guide RNA targets asequence within the vector which encodes the CRISPR C2c1 or C2c3effectorprotein, and thereby inactivates the effector protein's expression fromthat vector. Thus the target sequence in the vector must be capable ofinactivating expression. Suitable target sequences can be, for instance,near to or within the translational start codon for the C2c1 or C2c3effector protein coding sequence, in a non-coding sequence in thepromoter driving expression of the non-coding RNA elements, within thepromoter driving expression of the C2c1 or C2c3 effector protein gene,within 100 bp of the ATG translational start codon in the Cas codingsequence, and/or within the inverted terminal repeat (iTR) of a viraldelivery vector, e.g., in the AAV genome. A double stranded break nearthis region can induce a frame shift in the Cas coding sequence, causinga loss of protein expression. An alternative target sequence for the“self-inactivating” guide RNA would aim to edit/inactivate regulatoryregions/sequences needed for the expression of the CRISPR-C2c1 or C2c3system or for the stability of the vector. For instance, if the promoterfor the Cas coding sequence is disrupted then transcription can beinhibited or prevented. Similarly, if a vector includes sequences forreplication, maintenance or stability then it is possible to targetthese. For instance, in a AAV vector a useful target sequence is withinthe iTR. Other useful sequences to target can be promoter sequences,polyadenylation sites, etc.

Furthermore, if the guide RNAs are expressed in array format, the“self-inactivating” guide RNAs that target both promoters simultaneouslywill result in the excision of the intervening nucleotides from withinthe CRISPR-Cas expression construct, effectively leading to its completeinactivation. Similarly, excision of the intervening nucleotides willresult where the guide RNAs target both ITRs, or targets two or moreother CRISPR-Cas components simultaneously. Self-inactivation asexplained herein is applicable, in general, with CRISPR-Cas systems inorder to provide regulation of the CRISPR-Cas. For example,self-inactivation as explained herein may be applied to the CRISPRrepair of mutations, for example expansion disorders, as explainedherein. As a result of this self-inactivation, CRISPR repair is onlytransiently active.

Addition of non-targeting nucleotides to the 5′ end (e.g. 1-10nucleotides, preferably 1-5 nucleotides) of the “self-inactivating”guide RNA can be used to delay its processing and/or modify itsefficiency as a means of ensuring editing at the targeted genomic locusprior to CRISPR-Cas shutdown.

In one aspect of the self-inactivating AAV-CRISPR-Cas system, plasmidsthat co-express one or more guide RNA targeting genomic sequences ofinterest (e.g. 1-2, 1-5, 1-10, 1-15, 1-20, 1-30) may be established with“self-inactivating” guide RNAs that target an SpCas9 sequence at or nearthe engineered ATG start site (e.g. within 5 nucleotides, within 15nucleotides, within 30 nucleotides, within 50 nucleotides, within 100nucleotides). A regulatory sequence in the U6 promoter region can alsobe targeted with an guide RNA. The U6-driven guide RNAs may be designedin an array format such that multiple guide RNA sequences can besimultaneously released. When first delivered into target tissue/cells(left cell) guide RNAs begin to accumulate while Cas levels rise in thenucleus. Cas complexes with all of the guide RNAs to mediate genomeediting and self-inactivation of the CRISPR-Cas plasmids.

One aspect of a self-inactivating CRISPR-Cas system is expression ofsingly or in tandem array format from 1 up to 4 or more different guidesequences; e.g. up to about 20 or about 30 guides sequences. Eachindividual self inactivating guide sequence may target a differenttarget. Such may be processed from, e.g. one chimeric pol3 transcript.Pol3 promoters such as U6 or H1 promoters may be used. Pol2 promoterssuch as those mentioned throughout herein. Inverted terminal repeat(iTR) sequences may flank the Pol3 promoter—guide RNA(s)-Pol2promoter-Cas.

One aspect of a tandem array transcript is that one or more guide(s)edit the one or more target(s) while one or more self inactivatingguides inactivate the CRISPR-Cas system. Thus, for example, thedescribed CRISPR-Cas system for repairing expansion disorders may bedirectly combined with the self-inactivating CRISPR-Cas system describedherein. Such a system may, for example, have two guides directed to thetarget region for repair as well as at least a third guide directed toself-inactivation of the CRISPR-Cas. Reference is made to ApplicationSer. No. PCT/US2014/069897, entitled “Compositions And Methods Of Use OfCrispr-Cas Systems In Nucleotide Repeat Disorders,” published Dec. 12,2014 as WO/2015/089351.

The guideRNA may be a control guide. For example it may be engineered totarget a nucleic acid sequence encoding the CRISPR Enzyme itself, asdescribed in US2015232881A1, the disclosure of which is herebyincorporated by reference. In some embodiments, a system or compositionmay be provided with just the guideRNA engineered to target the nucleicacid sequence encoding the CRISPR Enzyme. In addition, the system orcomposition may be provided with the guideRNA engineered to target thenucleic acid sequence encoding the CRISPR Enzyme, as well as nucleicacid sequence encoding the CRISPR Enzyme and, optionally a second guideRNA and, further optionally, a repair template. The second guideRNA maybe the primary target of the CRISPR system or composition (such atherapeutic, diagnostic, knock out etc. as defined herein). In this way,the system or composition is self-inactivating. This is exemplified inrelation to Cas9 in US2015232881A1 (also published as WO2015070083 (A1)referenced elsewhere herein, and may be extrapolated to C2c1 or C2c3.Enzymes according to the invention used in a multiplex (tandem)targeting approach

The inventors have shown that CRISPR enzymes as defined herein canemploy more than one RNA guide without losing activity. This enables theuse of the CRISPR enzymes, systems or complexes as defined herein fortargeting multiple DNA targets, genes or gene loci, with a singleenzyme, system or complex as defined herein. The guide RNAs may betandemly arranged, optionally separated by a nucleotide sequence such asa direct repeat as defined herein. The position of the different guideRNAs is the tandem does not influence the activity. It is noted that theterms “CRISPR-Cas system”, “CRISP-Cas complex” “CRISPR complex” and“CRISPR system” are used interchangeably. Also the terms “CRISPRenzyme”, “Cas enzyme”, or “CRISPR-Cas enzyme”, can be usedinterchangeably. In preferred embodiments, said CRISPR enzyme, CRISP-Casenzyme or Cas enzyme is C2c1 or C2c3, or any one of the modified ormutated variants thereof described herein elsewhere.

In one aspect, the invention provides a non-naturally occurring orengineered CRISPR enzyme, preferably a class 2 CRISPR enzyme, preferablya Type V or VI CRISPR enzyme as described herein, such as withoutlimitation C2c1 or C2c3 as described herein elsewhere, used for tandemor multiplex targeting. It is to be understood that any of the CRISPR(or CRISPR-Cas or Cas) enzymes, complexes, or systems according to theinvention as described herein elsewhere may be used in such an approach.Any of the methods, products, compositions and uses as described hereinelsewhere are equally applicable with the multiplex or tandem targetingapproach further detailed below. By means of further guidance, thefollowing particular aspects and embodiments are provided.

In one aspect, the invention provides for the use of a C2c1 or C2c3enzyme, complex or system as defined herein for targeting multiple geneloci. In one embodiment, this can be established by using multiple(tandem or multiplex) guide RNA (gRNA) sequences.

In one aspect, the invention provides methods for using one or moreelements of a C2c1 or C2c3 enzyme, complex or system as defined hereinfor tandem or multiplex targeting, wherein said CRISP system comprisesmultiple guide RNA sequences. Preferably, said gRNA sequences areseparated by a nucleotide sequence, such as a direct repeat as definedherein elsewhere.

The C2c1 or C2c3 enzyme, system or complex as defined herein provides aneffective means for modifying multiple target polynucleotides. The C2c1or C2c3 enzyme, system or complex as defined herein has a wide varietyof utility including modifying (e.g., deleting, inserting,translocating, inactivating, activating) one or more targetpolynucleotides in a multiplicity of cell types. As such the C2c1 orC2c3 enzyme, system or complex as defined herein of the invention has abroad spectrum of applications in, e.g., gene therapy, drug screening,disease diagnosis, and prognosis, including targeting multiple gene lociwithin a single CRISPR system.

In one aspect, the invention provides a C2c1 or C2c3 enzyme, system orcomplex as defined herein, i.e. a C2c1 or C2c3 CRISPR-Cas complex havinga C2c1 or C2c3 protein having at least one destabilization domainassociated therewith, and multiple guide RNAs that target multiplenucleic acid molecules such as DNA molecules, whereby each of saidmultiple guide RNAs specifically targets its corresponding nucleic acidmolecule, e.g., DNA molecule. Each nucleic acid molecule target, e.g.,DNA molecule can encode a gene product or encompass a gene locus. Usingmultiple guide RNAs hence enables the targeting of multiple gene loci ormultiple genes. In some embodiments the C2c1 or C2c3 enzyme may cleavethe DNA molecule encoding the gene product. In some embodimentsexpression of the gene product is altered. The C2c1 or C2c3 protein andthe guide RNAs do not naturally occur together. The inventioncomprehends the guide RNAs comprising tandemly arranged guide sequences.The invention further comprehends coding sequences for the C2c1 or C2c3protein being codon optimized for expression in a eukaryotic cell. In apreferred embodiment the eukaryotic cell is a mammalian cell, a plantcell or a yeast cell and in a more preferred embodiment the mammaliancell is a human cell. Expression of the gene product may be decreased.The C2c1 or C2c3 enzyme may form part of a CRISPR system or complex,which further comprises tandemly arranged guide RNAs (gRNAs) comprisinga series of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 25, 25, 30, or more than 30guide sequences, each capable of specifically hybridizing to a targetsequence in a genomic locus of interest in a cell. In some embodiments,the functional C2c1 or C2c3 CRISPR system or complex binds to themultiple target sequences. In some embodiments, the functional CRISPRsystem or complex may edit the multiple target sequences, e.g., thetarget sequences may comprise a genomic locus, and in some embodimentsthere may be an alteration of gene expression. In some embodiments, thefunctional CRISPR system or complex may comprise further functionaldomains. In some embodiments, the invention provides a method foraltering or modifying expression of multiple gene products. The methodmay comprise introducing into a cell containing said target nucleicacids, e.g., DNA molecules, or containing and expressing target nucleicacid, e.g., DNA molecules; for instance, the target nucleic acids mayencode gene products or provide for expression of gene products (e.g.,regulatory sequences).

In preferred embodiments the CRISPR enzyme used for multiplex targetingis C2c1 or C2c3, or the CRISPR system or complex comprises C2c1 or C2c3.In some embodiments, the CRISPR enzyme used for multiplex targeting isAacC2c1, or the CRISPR system or complex used for multiplex targetingcomprises an AacC2c1. In some embodiments, the CRISPR enzyme is anAacC2c1, or the CRISPR system or complex comprises AacC2c1. In someembodiments, the C2c1 enzyme used for multiplex targeting cleaves bothstrands of DNA to produce a double strand break (DSB). In someembodiments, the CRISPR enzyme used for multiplex targeting is anickase. In some embodiments, the C2c1 or C2c3 enzyme used for multiplextargeting is a dual nickase. In some embodiments, the C2c1 or C2c3enzyme used for multiplex targeting is a C2c1 or C2c3 enzyme such as aC2c1 or C2c3 enzyme as defined herein elsewhere.

In some general embodiments, the C2c1 or C2c3 enzyme used for multiplextargeting is associated with one or more functional domains. In somemore specific embodiments, the CRISPR enzyme used for multiplextargeting is a dead-C2c1 or dead-C2c3 as defined herein elsewhere.

In an aspect, the present invention provides a means for delivering theC2c1 or C2c3 enzyme, system or complex for use in multiple targeting asdefined herein or the polynucleotides defined herein. Non-limitingexamples of such delivery means are e.g. particle(s) deliveringcomponent(s) of the complex, vector(s) comprising the polynucleotide(s)discussed herein (e.g., encoding the CRISPR enzyme, providing thenucleotides encoding the CRISPR complex). In some embodiments, thevector may be a plasmid or a viral vector such as AAV, or lentivirus.Transient transfection with plasmids, e.g., into HEK cells may beadvantageous, especially given the size limitations of AAV and thatwhile C2c1 or C2c3 fits into AAV, one may reach an upper limit withadditional guide RNAs.

Also provided is a model that constitutively expresses the C2c1 or C2c3enzyme, complex or system as used herein for use in multiplex targeting.The organism may be transgenic and may have been transfected with thepresent vectors or may be the offspring of an organism so transfected.In a further aspect, the present invention provides compositionscomprising the CRISPR enzyme, system and complex as defined herein orthe polynucleotides or vectors described herein. Also provides are C2c1or C2c3 CRISPR systems or complexes comprising multiple guide RNAs,preferably in a tandemly arranged format. Said different guide RNAs maybe separated by nucleotide sequences such as direct repeats.

Also provided is a method of treating a subject, e.g., a subject in needthereof, comprising inducing gene editing by transforming the subjectwith the polynucleotide encoding the C2c1 or C2c3 CRISPR system orcomplex or any of polynucleotides or vectors described herein andadministering them to the subject. A suitable repair template may alsobe provided, for example delivered by a vector comprising said repairtemplate. Also provided is a method of treating a subject, e.g., asubject in need thereof, comprising inducing transcriptional activationor repression of multiple target gene loci by transforming the subjectwith the polynucleotides or vectors described herein, wherein saidpolynucleotide or vector encodes or comprises the C2c1 or C2c3 enzyme,complex or system comprising multiple guide RNAs, preferably tandemlyarranged. Where any treatment is occurring ex vivo, for example in acell culture, then it will be appreciated that the term ‘subject’ may bereplaced by the phrase “cell or cell culture.”

Compositions comprising C2c1 or C2c3 enzyme, complex or systemcomprising multiple guide RNAs, preferably tandemly arranged, or thepolynucleotide or vector encoding or comprising said C2c1 or C2c3enzyme, complex or system comprising multiple guide RNAs, preferablytandemly arranged, for use in the methods of treatment as defined hereinelsewhere are also provided. A kit of parts may be provided includingsuch compositions. Use of said composition in the manufacture of amedicament for such methods of treatment are also provided. Use of aC2c1 or C2c3 CRISPR system in screening is also provided by the presentinvention, e.g., gain of function screens. Cells which are artificiallyforced to overexpress a gene are be able to down regulate the gene overtime (re-establishing equilibrium) e.g. by negative feedback loops. Bythe time the screen starts the unregulated gene might be reduced again.Using an inducible C2c1 or C2c3 activator allows one to inducetranscription right before the screen and therefore minimizes the chanceof false negative hits. Accordingly, by use of the instant invention inscreening, e.g., gain of function screens, the chance of false negativeresults may be minimized.

In one aspect, the invention provides an engineered, non-naturallyoccurring CRISPR system comprising a C2c1 or C2c3 protein and multipleguide RNAs that each specifically target a DNA molecule encoding a geneproduct in a cell, whereby the multiple guide RNAs each target theirspecific DNA molecule encoding the gene product and the C2c1 or C2c3protein cleaves the target DNA molecule encoding the gene product,whereby expression of the gene product is altered; and, wherein theCRISPR protein and the guide RNAs do not naturally occur together. Theinvention comprehends the multiple guide RNAs comprising multiple guidesequences, preferably separated by a nucleotide sequence such as adirect repeat. In an embodiment of the invention the CRISPR protein is atype V or VI CRISPR-Cas protein and in a more preferred embodiment theCRIPSR protein is a C2c1 or C2c3 protein. The invention furthercomprehends a C2c1 or C2c3 protein being codon optimized for expressionin a eukaryotic cell. In a preferred embodiment the eukaryotic cell is amammalian cell and in a more preferred embodiment the mammalian cell isa human cell. In a further embodiment of the invention, the expressionof the gene product is decreased.

In another aspect, the invention provides an engineered, non-naturallyoccurring vector system comprising one or more vectors comprising afirst regulatory element operably linked to the multiple C2c1 or C2c3CRISPR system guide RNAs that each specifically target a DNA moleculeencoding a gene product and a second regulatory element operably linkedcoding for a CRISPR protein. Both regulatory elements may be located onthe same vector or on different vectors of the system. The multipleguide RNAs target the multiple DNA molecules encoding the multiple geneproducts in a cell and the CRISPR protein may cleave the multiple DNAmolecules encoding the gene products (it may cleave one or both strandsor have substantially no nuclease activity), whereby expression of themultiple gene products is altered; and, wherein the CRISPR protein andthe multiple guide RNAs do not naturally occur together. In a preferredembodiment the CRISPR protein is C2c1 or C2c3 protein, optionally codonoptimized for expression in a eukaryotic cell. In a preferred embodimentthe eukaryotic cell is a mammalian cell, a plant cell or a yeast celland in a more preferred embodiment the mammalian cell is a human cell.In a further embodiment of the invention, the expression of each of themultiple gene products is altered, preferably decreased.

In one aspect, the invention provides a vector system comprising one ormore vectors. In some embodiments, the system comprises: (a) a firstregulatory element operably linked to a direct repeat sequence and oneor more insertion sites for inserting one or more guide sequences up- ordownstream (whichever applicable) of the direct repeat sequence, whereinwhen expressed, the one or more guide sequence(s) direct(s)sequence-specific binding of the CRISPR complex to the one or moretarget sequence(s) in a eukaryotic cell, wherein the CRISPR complexcomprises a C2c1 or C2c3 enzyme complexed with the one or more guidesequence(s) that is hybridized to the one or more target sequence(s);and (b) a second regulatory element operably linked to an enzyme-codingsequence encoding said C2c1 or C2c3 enzyme, preferably comprising atleast one nuclear localization sequence and/or at least one NES; whereincomponents (a) and (b) are located on the same or different vectors ofthe system. In some embodiments, component (a) further comprises two ormore guide sequences operably linked to the first regulatory element,wherein when expressed, each of the two or more guide sequences directsequence specific binding of a C2c1 or C2c3 CRISPR complex to adifferent target sequence in a eukaryotic cell. In some embodiments, theCRISPR complex comprises one or more nuclear localization sequencesand/or one or more NES of sufficient strength to drive accumulation ofsaid C2c1 or C2c3 CRISPR complex in a detectable amount in or out of thenucleus of a eukaryotic cell. In some embodiments, the first regulatoryelement is a polymerase III promoter. In some embodiments, the secondregulatory element is a polymerase II promoter. In some embodiments,each of the guide sequences is at least 16, 17, 18, 19, 20, 25nucleotides, or between 16-30, or between 16-25, or between 16-20nucleotides in length.

Recombinant expression vectors can comprise the polynucleotides encodingthe C2c1 or C2c3 enzyme, system or complex for use in multiple targetingas defined herein in a form suitable for expression of the nucleic acidin a host cell, which means that the recombinant expression vectorsinclude one or more regulatory elements, which may be selected on thebasis of the host cells to be used for expression, that isoperatively-linked to the nucleic acid sequence to be expressed. Withina recombinant expression vector, “operably linked” is intended to meanthat the nucleotide sequence of interest is linked to the regulatoryelement(s) in a manner that allows for expression of the nucleotidesequence (e.g., in an in vitro transcription/translation system or in ahost cell when the vector is introduced into the host cell).

In some embodiments, a host cell is transiently or non-transientlytransfected with one or more vectors comprising the polynucleotidesencoding the C2c1 or C2c3 enzyme, system or complex for use in multipletargeting as defined herein. In some embodiments, a cell is transfectedas it naturally occurs in a subject. In some embodiments, a cell that istransfected is taken from a subject. In some embodiments, the cell isderived from cells taken from a subject, such as a cell line. A widevariety of cell lines for tissue culture are known in the art andexemplified herein elsewhere. Cell lines are available from a variety ofsources known to those with skill in the art (see, e.g., the AmericanType Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, acell transfected with one or more vectors comprising the polynucleotidesencoding the C2c1 or C2c3 enzyme, system or complex for use in multipletargeting as defined herein is used to establish a new cell linecomprising one or more vector-derived sequences. In some embodiments, acell transiently transfected with the components of a C2c1 or C2c3CRISPR system or complex for use in multiple targeting as describedherein (such as by transient transfection of one or more vectors, ortransfection with RNA), and modified through the activity of a C2c1 orC2c3 CRISPR system or complex, is used to establish a new cell linecomprising cells containing the modification but lacking any otherexogenous sequence. In some embodiments, cells transiently ornon-transiently transfected with one or more vectors comprising thepolynucleotides encoding the C2c1 or C2c3 enzyme, system or complex foruse in multiple targeting as defined herein, or cell lines derived fromsuch cells are used in assessing one or more test compounds.

The term “regulatory element” is as defined herein elsewhere.

Advantageous vectors include lentiviruses and adeno-associated viruses,and types of such vectors can also be selected for targeting particulartypes of cells.

In one aspect, the invention provides a eukaryotic host cell comprising(a) a first regulatory element operably linked to a direct repeatsequence and one or more insertion sites for inserting one or more guideRNA sequences up- or downstream (whichever applicable) of the directrepeat sequence, wherein when expressed, the guide sequence(s) direct(s)sequence-specific binding of the C2c1 or C2c3 CRISPR complex to therespective target sequence(s) in a eukaryotic cell, wherein the C2c1 orC2c3 CRISPR complex comprises a C2c1 or C2c3 enzyme complexed with theone or more guide sequence(s) that is hybridized to the respectivetarget sequence(s); and/or (b) a second regulatory element operablylinked to an enzyme-coding sequence encoding said C2c1 or C2c3 enzymecomprising preferably at least one nuclear localization sequence and/orNES. In some embodiments, the host cell comprises components (a) and(b). In some embodiments, component (a), component (b), or components(a) and (b) are stably integrated into a genome of the host eukaryoticcell. In some embodiments, component (a) further comprises two or moreguide sequences operably linked to the first regulatory element, andoptionally separated by a direct repeat, wherein when expressed, each ofthe two or more guide sequences direct sequence specific binding of aC2c1 or C2c3 CRISPR complex to a different target sequence in aeukaryotic cell. In some embodiments, the C2c1 or C2c3 enzyme comprisesone or more nuclear localization sequences and/or nuclear exportsequences or NES of sufficient strength to drive accumulation of saidCRISPR enzyme in a detectable amount in and/or out of the nucleus of aeukaryotic cell.

In some embodiments, the C2c1 or C2c3 enzyme is a type V or VI CRISPRsystem enzyme. In some embodiments, the C2c1 enzyme is an AacC2c1enzyme. In some embodiments, the C2c1 enzyme is derived fromAlicyclobacillus acidoterrestris (e.g., ATCC 49025), Alicyclobacilluscontaminans (e.g., DSM 17975), Desulfovibrio inopinatus (e.g., DSM10711), Desulfonatronum thiodismutans (e.g., strain MLF-1), Opitutaceaebacterium TAV5, Tuberibacillus calidus (e.g., DSM 17572), Bacillusthermoamylovorans (e.g., strain B4166), Brevibacillus sp. CF112,Bacillus sp. NSP2.1, Desulfatirhabdium butyrativorans (e.g., DSM 18734),Alicyclobacillus herbarius (e.g., DSM 13609), Citrobacter freundii(e.g., ATCC 8090), Brevibacillus agri (e.g., BAB-2500), Methylobacteriumnodulans (e.g., ORS 2060), and may include further alterations ormutations of the C2c1 as defined herein elsewhere, and can be a chimericC2c1. In some embodiments, the C2c1 or C2c3 enzyme is codon-optimizedfor expression in a eukaryotic cell. In some embodiments, the CRISPRenzyme directs cleavage of one or two strands at the location of thetarget sequence. In some embodiments, the first regulatory element is apolymerase III promoter. In some embodiments, the second regulatoryelement is a polymerase II promoter. In some embodiments, the one ormore guide sequence(s) is (are each) at least 16, 17, 18, 19, 20, 25nucleotides, or between 16-30, or between 16-25, or between 16-20nucleotides in length. When multiple guide RNAs are used, they arepreferably separated by a direct repeat sequence. In an aspect, theinvention provides a non-human eukaryotic organism; preferably amulticellular eukaryotic organism, comprising a eukaryotic host cellaccording to any of the described embodiments. In other aspects, theinvention provides a eukaryotic organism; preferably a multicellulareukaryotic organism, comprising a eukaryotic host cell according to anyof the described embodiments. The organism in some embodiments of theseaspects may be an animal; for example a mammal. Also, the organism maybe an arthropod such as an insect. The organism also may be a plant.Further, the organism may be a fungus.

In one aspect, the invention provides a kit comprising one or more ofthe components described herein. In some embodiments, the kit comprisesa vector system and instructions for using the kit. In some embodiments,the vector system comprises (a) a first regulatory element operablylinked to a direct repeat sequence and one or more insertion sites forinserting one or more guide sequences up- or downstream (whicheverapplicable) of the direct repeat sequence, wherein when expressed, theguide sequence directs sequence-specific binding of a C2c1 or C2c3CRISPR complex to a target sequence in a eukaryotic cell, wherein theC2c1 CRISPR complex comprises a C2c1 or C2c3 enzyme complexed with theguide sequence that is hybridized to the target sequence; and/or (b) asecond regulatory element operably linked to an enzyme-coding sequenceencoding said C2c1 or C2c3 enzyme comprising a nuclear localizationsequence. In some embodiments, the kit comprises components (a) and (b)located on the same or different vectors of the system. In someembodiments, component (a) further comprises two or more guide sequencesoperably linked to the first regulatory element, wherein when expressed,each of the two or more guide sequences direct sequence specific bindingof a CRISPR complex to a different target sequence in a eukaryotic cell.In some embodiments, the C2c1 or C2c3 enzyme comprises one or morenuclear localization sequences of sufficient strength to driveaccumulation of said CRISPR enzyme in a detectable amount in the nucleusof a eukaryotic cell. In some embodiments, the CRISPR enzyme is a type Vor VI CRISPR system enzyme. In some embodiments, the CRISPR enzyme is aC2c1 or C2c3 enzyme. In some embodiments, the C2c1 enzyme is derivedfrom Alicyclobacillus acidoterrestris (e.g., ATCC 49025),Alicyclobacillus contaminans (e.g., DSM 17975), Desulfovibrio inopinatus(e.g., DSM 10711), Desulfonatronum thiodismutans (e.g., strain MLF-1),Opitutaceae bacterium TAV5, Tuberibacillus calidus (e.g., DSM 17572),Bacillus thermoamylovorans (e.g., strain B4166), Brevibacillus sp.CF112, Bacillus sp. NSP2.1, Desulfatirhabdium butyrativorans (e.g., DSM18734), Alicyclobacillus herbarius (e.g., DSM 13609), Citrobacterfreundii (e.g., ATCC 8090), Brevibacillus agri (e.g., BAB-2500),Methylobacterium nodulans (e.g., ORS 2060) (e.g., modified to have or beassociated with at least one destabilizing domain (“DD”), and mayinclude further alteration or mutation of the C2c1, and can be achimeric C2c1. In some embodiments, the DD-CRISPR enzyme iscodon-optimized for expression in a eukaryotic cell. In someembodiments, the DD-CRISPR enzyme directs cleavage of one or two strandsat the location of the target sequence. In some embodiments, theDD-CRISPR enzyme lacks or substantially DNA strand cleavage activity(e.g., no more than 5% nuclease activity as compared with a wild typeenzyme or enzyme not having the mutation or alteration that decreasesnuclease activity). In some embodiments, the first regulatory element isa polymerase III promoter. In some embodiments, the second regulatoryelement is a polymerase II promoter. In some embodiments, the guidesequence is at least 16, 17, 18, 19, 20, 25 nucleotides, or between16-30, or between 16-25, or between 16-20 nucleotides in length.

In one aspect, the invention provides a method of modifying multipletarget polynucleotides in a host cell such as a eukaryotic cell. In someembodiments, the method comprises allowing a C2c1 or C2c3 CRISPR complexto bind to multiple target polynucleotides, e.g., to effect cleavage ofsaid multiple target polynucleotides, thereby modifying multiple targetpolynucleotides, wherein the C2c1 or C2c3 CRISPR complex comprises aC2c1 or C2c3 enzyme complexed with multiple guide sequences each of thebeing hybridized to a specific target sequence within said targetpolynucleotide, wherein said multiple guide sequences are linked to adirect repeat sequence. In some embodiments, said cleavage comprisescleaving one or two strands at the location of each of the targetsequence by said C2c1 or C2c3 enzyme. In some embodiments, said cleavageresults in decreased transcription of the multiple target genes. In someembodiments, the method further comprises repairing one or more of saidcleaved target polynucleotide by homologous recombination with anexogenous template polynucleotide, wherein said repair results in amutation comprising an insertion, deletion, or substitution of one ormore nucleotides of one or more of said target polynucleotides. In someembodiments, said mutation results in one or more amino acid changes ina protein expressed from a gene comprising one or more of the targetsequence(s). In some embodiments, the method further comprisesdelivering one or more vectors to said eukaryotic cell, wherein the oneor more vectors drive expression of one or more of: the C2c1 or C2c3enzyme and the multiple guide RNA sequence linked to a direct repeatsequence. In some embodiments, said vectors are delivered to theeukaryotic cell in a subject. In some embodiments, said modifying takesplace in said eukaryotic cell in a cell culture. In some embodiments,the method further comprises isolating said eukaryotic cell from asubject prior to said modifying. In some embodiments, the method furthercomprises returning said eukaryotic cell and/or cells derived therefromto said subject.

In one aspect, the invention provides a method of modifying expressionof multiple polynucleotides in a eukaryotic cell. In some embodiments,the method comprises allowing a C2c1 or C2c3 CRISPR complex to bind tomultiple polynucleotides such that said binding results in increased ordecreased expression of said polynucleotides; wherein the C2c1 or C2c3CRISPR complex comprises a C2c1 or C2c3 enzyme complexed with multipleguide sequences each specifically hybridized to its own target sequencewithin said polynucleotide, wherein said guide sequences are linked to adirect repeat sequence. In some embodiments, the method furthercomprises delivering one or more vectors to said eukaryotic cells,wherein the one or more vectors drive expression of one or more of: theC2c1 or C2c3 enzyme and the multiple guide sequences linked to thedirect repeat sequences.

In one aspect, the invention provides a recombinant polynucleotidecomprising multiple guide RNA sequences up- or downstream (whicheverapplicable) of a direct repeat sequence, wherein each of the guidesequences when expressed directs sequence-specific binding of a C2c1 orC2c3 CRISPR complex to its corresponding target sequence present in aeukaryotic cell. In some embodiments, the target sequence is a viralsequence present in a eukaryotic cell. In some embodiments, the targetsequence is a proto-oncogene or an oncogene.

Aspects of the invention encompass a non-naturally occurring orengineered composition that may comprise a guide RNA (gRNA) comprising aguide sequence capable of hybridizing to a target sequence in a genomiclocus of interest in a cell and a C2c1 or C2c3 enzyme as defined hereinthat may comprise at least one or more nuclear localization sequences.

An aspect of the invention encompasses methods of modifying a genomiclocus of interest to change gene expression in a cell by introducinginto the cell any of the compositions described herein.

An aspect of the invention is that the above elements are comprised in asingle composition or comprised in individual compositions. Thesecompositions may advantageously be applied to a host to elicit afunctional effect on the genomic level.

As used herein, the term “guide RNA” or “gRNA” has the leaning as usedherein elsewhere and comprises any polynucleotide sequence havingsufficient complementarity with a target nucleic acid sequence tohybridize with the target nucleic acid sequence and directsequence-specific binding of a nucleic acid-targeting complex to thetarget nucleic acid sequence. Each gRNA may be designed to includemultiple binding recognition sites (e.g., aptamers) specific to the sameor different adapter protein. Each gRNA may be designed to bind to thepromoter region −1000-+1 nucleic acids upstream of the transcriptionstart site (i.e. TSS), preferably −200 nucleic acids. This positioningimproves functional domains which affect gene activation (e.g.,transcription activators) or gene inhibition (e.g., transcriptionrepressors). The modified gRNA may be one or more modified gRNAstargeted to one or more target loci (e.g., at least 1 gRNA, at least 2gRNA, at least 5 gRNA, at least 10 gRNA, at least 20 gRNA, at least 30 gRNA, at least 50 gRNA) comprised in a composition. Said multiple gRNAsequences can be tandemly arranged and are preferably separated by adirect repeat.

Thus, gRNA, the CRISPR enzyme as defined herein may each individually becomprised in a composition and administered to a host individually orcollectively. Alternatively, these components may be provided in asingle composition for administration to a host. Administration to ahost may be performed via viral vectors known to the skilled person ordescribed herein for delivery to a host (e.g., lentiviral vector,adenoviral vector, AAV vector). As explained herein, use of differentselection markers (e.g., for lentiviral gRNA selection) andconcentration of gRNA (e.g., dependent on whether multiple gRNAs areused) may be advantageous for eliciting an improved effect. On the basisof this concept, several variations are appropriate to elicit a genomiclocus event, including DNA cleavage, gene activation, or genedeactivation. Using the provided compositions, the person skilled in theart can advantageously and specifically target single or multiple lociwith the same or different functional domains to elicit one or moregenomic locus events. The compositions may be applied in a wide varietyof methods for screening in libraries in cells and functional modelingin vivo (e.g., gene activation of lincRNA and identification offunction; gain-of-function modeling; loss-of-function modeling; the usethe compositions of the invention to establish cell lines and transgenicanimals for optimization and screening purposes).

The current invention comprehends the use of the compositions of thecurrent invention to establish and utilize conditional or inducibleCRISPR transgenic cell/animals; see. e.g., Platt et al., Cell (2014),159(2): 440-455, or PCT patent publications cited herein, such as WO2014/093622 (PCT/US2013/074667). For example, cells or animals such asnon-human animals, e.g., vertebrates or mammals, such as rodents, e.g.,mice, rats, or other laboratory or field animals, e.g., cats, dogs,sheep, etc., may be ‘knock-in’ whereby the animal conditionally orinducibly expresses C2c1 or C2c3 akin to Platt et al. The target cell oranimal thus comprises the CRISPR enzyme (e.g., C2c1 or C2c3)conditionally or inducibly (e.g., in the form of Cre dependentconstructs), on expression of a vector introduced into the target cell,the vector expresses that which induces or gives rise to the conditionof the CRISPR enzyme (e.g., C2c1 or C2c3) expression in the target cell.By applying the teaching and compositions as defined herein with theknown method of creating a CRISPR complex, inducible genomic events arealso an aspect of the current invention. Examples of such inducibleevents have been described herein elsewhere.

In some embodiments, phenotypic alteration is preferably the result ofgenome modification when a genetic disease is targeted, especially inmethods of therapy and preferably where a repair template is provided tocorrect or alter the phenotype.

In some embodiments diseases that may be targeted include thoseconcerned with disease-causing splice defects.

In some embodiments, cellular targets include HemopoieticStem/Progenitor Cells (CD34+); Human T cells; and Eye (retinalcells)—for example photoreceptor precursor cells.

In some embodiments Gene targets include: Human Beta Globin—HBB (fortreating Sickle Cell Anemia, including by stimulating gene-conversion(using closely related HBD gene as an endogenous template)); CD3(T-Cells); and CEP920-retina (eye).

In some embodiments disease targets also include: cancer; Sickle CellAnemia (based on a point mutation); HBV, HIV; Beta-Thalassemia; andophthalmic or ocular disease—for example Leber Congenital Amaurosis(LCA)-causing Splice Defect.

In some embodiments delivery methods include: Cationic Lipid Mediated“direct” delivery of Enzyme-Guide complex (RiboNucleoProtein) andelectroporation of plasmid DNA.

Methods, products and uses described herein may be used fornon-therapeutic purposes. Furthermore, any of the methods describedherein may be applied in vitro and ex vivo.

In an aspect, provided is a non-naturally occurring or engineeredcomposition comprising:

I. two or more CRISPR-Cas system polynucleotide sequences comprising

(a) a first guide sequence capable of hybridizing to a first targetsequence in a polynucleotide locus,

(b) a second guide sequence capable of hybridizing to a second targetsequence in a polynucleotide locus,

(c) a direct repeat sequence,

and

II. a C2c1 enzyme or a second polynucleotide sequence encoding it or aC2c3 enzyme or a second polynucleotide sequence encoding it,

wherein when transcribed, the first and the second guide sequencesdirect sequence-specific binding of a first and a second C2c1 or C2c3CRISPR complex to the first and second target sequences respectively,

wherein the first CRISPR complex comprises the C2c1 enzyme or the C2c3enzyme complexed with the first guide sequence that is hybridizable tothe first target sequence,

wherein the second CRISPR complex comprises the C2c1 enzyme or the C2c3enzyme complexed with the second guide sequence that is hybridizable tothe second target sequence, and

wherein the first guide sequence directs cleavage of one strand of theDNA duplex near the first target sequence and the second guide sequencedirects cleavage of the other strand near the second target sequenceinducing a double strand break, thereby modifying the organism or thenon-human or non-animal organism. Similarly, compositions comprisingmore than two guide RNAs can be envisaged e.g. each specific for onetarget, and arranged tandemly in the composition or CRISPR system orcomplex as described herein.

In another embodiment, the C2c1 or C2c3 is delivered into the cell as aprotein. In another and particularly preferred embodiment, the C2c1 orC2c3 is delivered into the cell as a protein or as a nucleotide sequenceencoding it. Delivery to the cell as a protein may include delivery of aRibonucleoprotein (RNP) complex, where the protein is complexed with themultiple guides.

In an aspect, host cells and cell lines modified by or comprising thecompositions, systems or modified enzymes of present invention areprovided, including stem cells, and progeny thereof.

In an aspect, methods of cellular therapy are provided, where, forexample, a single cell or a population of cells is sampled or cultured,wherein that cell or cells is or has been modified ex vivo as describedherein, and is then re-introduced (sampled cells) or introduced(cultured cells) into the organism. Stem cells, whether embryonic orinduce pluripotent or totipotent stem cells, are also particularlypreferred in this regard. But, of course, in vivo embodiments are alsoenvisaged.

Inventive methods can further comprise delivery of templates, such asrepair templates, which may be dsODN or ssODN, see below. Delivery oftemplates may be via the cotemporaneous or separate from delivery of anyor all the CRISPR enzyme or guide RNAs and via the same deliverymechanism or different. In some embodiments, it is preferred that thetemplate is delivered together with the guide RNAs and, preferably, alsothe CRISPR enzyme. An example may be an AAV vector where the CRISPRenzyme is C2c1 or C2c3, preferably AacC2c1.

Inventive methods can further comprise: (a) delivering to the cell adouble-stranded oligodeoxynucleotide (dsODN) comprising overhangscomplimentary to the overhangs created by said double strand break,wherein said dsODN is integrated into the locus of interest; or—(b)delivering to the cell a single-stranded oligodeoxynucleotide (ssODN),wherein said ssODN acts as a template for homology directed repair ofsaid double strand break. Inventive methods can be for the prevention ortreatment of disease in an individual, optionally wherein said diseaseis caused by a defect in said locus of interest. Inventive methods canbe conducted in vivo in the individual or ex vivo on a cell taken fromthe individual, optionally wherein said cell is returned to theindividual.

The invention also comprehends products obtained from using CRISPRenzyme or Cas enzyme or C2c1 enzyme or C2c3 enzyme or CRISPR-CRISPRenzyme or CRISPR-Cas system or CRISPR-C2c1 system or CRISPR-C2c3 systemfor use in tandem or multiple targeting as defined herein.

In one aspect, the invention provides kits containing any one or more ofthe elements disclosed in the above methods and compositions. In someembodiments, the kit comprises a vector system as taught herein andinstructions for using the kit. Elements may be provide individually orin combinations, and may be provided in any suitable container, such asa vial, a bottle, or a tube. In some embodiments, the kit includesinstructions in one or more languages, for example in more than onelanguage. The instructions may be specific to the applications andmethods described herein.

In some embodiments, a kit comprises one or more reagents for use in aprocess utilizing one or more of the elements described herein. Reagentsmay be provided in any suitable container. For example, a kit mayprovide one or more reaction or storage buffers. Reagents may beprovided in a form that is usable in a particular assay, or in a formthat requires addition of one or more other components before use (e.g.,in concentrate or lyophilized form). A buffer can be any buffer,including but not limited to a sodium carbonate buffer, a sodiumbicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, aHEPES buffer, and combinations thereof. In some embodiments, the bufferis alkaline. In some embodiments, the buffer has a pH from about 7 toabout 10. In some embodiments, the kit comprises one or moreoligonucleotides corresponding to a guide sequence for insertion into avector so as to operably link the guide sequence and a regulatoryelement. In some embodiments, the kit comprises a homologousrecombination template polynucleotide. In some embodiments, the kitcomprises one or more of the vectors and/or one or more of thepolynucleotides described herein. The kit may advantageously allows toprovide all elements of the systems of the invention.

In one aspect, the invention provides methods for using one or moreelements of a CRISPR system. The CRISPR complex of the inventionprovides an effective means for modifying a target polynucleotide. TheCRISPR complex of the invention has a wide variety of utility includingmodifying (e.g., deleting, inserting, translocating, inactivating,activating) a target polynucleotide in a multiplicity of cell types. Assuch the CRISPR complex of the invention has a broad spectrum ofapplications in, e.g., gene therapy, drug screening, disease diagnosis,and prognosis. An exemplary CRISPR complex comprises a CRISPR effectorprotein complexed with a guide sequence hybridized to a target sequencewithin the target polynucleotide. In certain embodiments, a directrepeat sequence is linked to the guide sequence.

In one embodiment, this invention provides a method of cleaving a targetpolynucleotide. The method comprises modifying a target polynucleotideusing a CRISPR complex that binds to the target polynucleotide andeffect cleavage of said target polynucleotide. Typically, the CRISPRcomplex of the invention, when introduced into a cell, creates a break(e.g., a single or a double strand break) in the genome sequence. Forexample, the method can be used to cleave a disease gene in a cell.

The break created by the CRISPR complex can be repaired by a repairprocesses such as the error prone non-homologous end joining (NHEJ)pathway or the high fidelity homology directed repair (HDR). Duringthese repair process, an exogenous polynucleotide template can beintroduced into the genome sequence. In some methods, the HDR process isused to modify genome sequence. For example, an exogenous polynucleotidetemplate comprising a sequence to be integrated flanked by an upstreamsequence and a downstream sequence is introduced into a cell. Theupstream and downstream sequences share sequence similarity with eitherside of the site of integration in the chromosome.

Where desired, a donor polynucleotide can be DNA, e.g., a DNA plasmid, abacterial artificial chromosome (BAC), a yeast artificial chromosome(YAC), a viral vector, a linear piece of DNA, a PCR fragment, a nakednucleic acid, or a nucleic acid complexed with a delivery vehicle suchas a liposome or poloxamer.

The exogenous polynucleotide template comprises a sequence to beintegrated (e.g., a mutated gene). The sequence for integration may be asequence endogenous or exogenous to the cell. Examples of a sequence tobe integrated include polynucleotides encoding a protein or a non-codingRNA (e.g., a microRNA). Thus, the sequence for integration may beoperably linked to an appropriate control sequence or sequences.Alternatively, the sequence to be integrated may provide a regulatoryfunction.

The upstream and downstream sequences in the exogenous polynucleotidetemplate are selected to promote recombination between the chromosomalsequence of interest and the donor polynucleotide. The upstream sequenceis a nucleic acid sequence that shares sequence similarity with thegenome sequence upstream of the targeted site for integration.Similarly, the downstream sequence is a nucleic acid sequence thatshares sequence similarity with the chromosomal sequence downstream ofthe targeted site of integration. The upstream and downstream sequencesin the exogenous polynucleotide template can have 75%, 80%, 85%, 90%,95%, or 100% sequence identity with the targeted genome sequence.Preferably, the upstream and downstream sequences in the exogenouspolynucleotide template have about 95%, 96%, 97%, 98%, 99%, or 100%sequence identity with the targeted genome sequence. In some methods,the upstream and downstream sequences in the exogenous polynucleotidetemplate have about 99% or 100% sequence identity with the targetedgenome sequence.

An upstream or downstream sequence may comprise from about 20 bp toabout 2500 bp, for example, about 50, 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900,2000, 2100, 2200, 2300, 2400, or 2500 bp. In some methods, the exemplaryupstream or downstream sequence have about 200 bp to about 2000 bp,about 600 bp to about 1000 bp, or more particularly about 700 bp toabout 1000 bp.

In some methods, the exogenous polynucleotide template may furthercomprise a marker. Such a marker may make it easy to screen for targetedintegrations. Examples of suitable markers include restriction sites,fluorescent proteins, or selectable markers. The exogenouspolynucleotide template of the invention can be constructed usingrecombinant techniques (see, for example, Sambrook et al., 2001 andAusubel et al., 1996).

In an exemplary method for modifying a target polynucleotide byintegrating an exogenous polynucleotide template, a double strandedbreak is introduced into the genome sequence by the CRISPR complex, thebreak is repaired via homologous recombination an exogenouspolynucleotide template such that the template is integrated into thegenome. The presence of a double-stranded break facilitates integrationof the template.

In other embodiments, this invention provides a method of modifyingexpression of a polynucleotide in a eukaryotic cell. The methodcomprises increasing or decreasing expression of a target polynucleotideby using a CRISPR complex that binds to the polynucleotide.

In some methods, a target polynucleotide can be inactivated to effectthe modification of the expression in a cell. For example, upon thebinding of a CRISPR complex to a target sequence in a cell, the targetpolynucleotide is inactivated such that the sequence is not transcribed,the coded protein is not produced, or the sequence does not function asthe wild-type sequence does. For example, a protein or microRNA codingsequence may be inactivated such that the protein is not produced.

In some methods, a control sequence can be inactivated such that it nolonger functions as a control sequence. As used herein, “controlsequence” refers to any nucleic acid sequence that effects thetranscription, translation, or accessibility of a nucleic acid sequence.Examples of a control sequence include, a promoter, a transcriptionterminator, and an enhancer are control sequences. The inactivatedtarget sequence may include a deletion mutation (i.e., deletion of oneor more nucleotides), an insertion mutation (i.e., insertion of one ormore nucleotides), or a nonsense mutation (i.e., substitution of asingle nucleotide for another nucleotide such that a stop codon isintroduced). In some methods, the inactivation of a target sequenceresults in “knockout” of the target sequence.

Exemplary Methods of Using of CRISPR Cas System

The invention provides a non-naturally occurring or engineeredcomposition, or one or more polynucleotides encoding components of saidcomposition, or vector or delivery systems comprising one or morepolynucleotides encoding components of said composition for use in amodifying a target cell in vivo, ex vivo or in vitro and, may beconducted in a manner alters the cell such that once modified theprogeny or cell line of the CRISPR modified cell retains the alteredphenotype. The modified cells and progeny may be part of amulti-cellular organism such as a plant or animal with ex vivo or invivo application of CRISPR system to desired cell types. The CRISPRinvention may be a therapeutic method of treatment. The therapeuticmethod of treatment may comprise gene or genome editing, or genetherapy.

Use of Inactivated CRISPR C2c1 or C2c3 Enzyme for Detection Methods Suchas FISH

In one aspect, the invention provides an engineered, non-naturallyoccurring CRISPR-Cas system comprising a catalytically inactivate Casprotein described herein, preferably an inactivate C2c1 (dC2c1) or aninactivate C2c3 (dC2c3), and use this system in detection methods suchas fluorescence in situ hybridization (FISH). dC2c1 or dC2c3 which lacksthe ability to produce DNA double-strand breaks may be fused with amarker, such as fluorescent protein, such as the enhanced greenfluorescent protein (eEGFP) and co-expressed with small guide RNAs totarget pericentric, centric and telomeric repeats in vivo. The dC2c1 ordC2c3 system can be used to visualize both repetitive sequences andindividual genes in the human genome. Such new applications of labelleddC2c1 or dC2c3 CRISPR-cas systems may be important in imaging cells andstudying the functional nuclear architecture, especially in cases with asmall nucleus volume or complex 3-D structures. (Chen B, Gilbert L A,Cimini B A, Schnitzbauer J, Zhang W, Li G W, Park J, Blackburn E H,Weissman J S, Qi L S, Huang B. 2013. Dynamic imaging of genomic loci inliving human cells by an optimized CRISPR/Cas system. Cell155(7):1479-91. doi: 10.1016/j.cell.2013.12.001.)

Modifying a Target with CRISPR Cas System or Complex (e.g.,C2c1/C2c3-RNA Complex)

In one aspect, the invention provides for methods of modifying a targetpolynucleotide in a eukaryotic cell, which may be in vivo, ex vivo or invitro. In some embodiments, the method comprises sampling a cell orpopulation of cells from a human or non-human animal, and modifying thecell or cells. Culturing may occur at any stage ex vivo. The cell orcells may even be re-introduced into the non-human animal or plant. Forre-introduced cells it is particularly preferred that the cells are stemcells.

In some embodiments, the method comprises allowing a CRISPR complex tobind to the target polynucleotide to effect cleavage of said targetpolynucleotide thereby modifying the target polynucleotide, wherein theCRISPR complex comprises a CRISPR effector protein complexed with aguide sequence hybridized or hybridizable to a target sequence withinsaid target polynucleotide.

In one aspect, the invention provides a method of modifying expressionof a polynucleotide in a eukaryotic cell. In some embodiments, themethod comprises allowing a CRISPR complex to bind to the polynucleotidesuch that said binding results in increased or decreased expression ofsaid polynucleotide; wherein the CRISPR complex comprises a CRISPReffector protein complexed with a guide sequence hybridized orhybridizable to a target sequence within said polynucleotide. Similarconsiderations and conditions apply as above for methods of modifying atarget polynucleotide. In fact, these sampling, culturing andre-introduction options apply across the aspects of the presentinvention.

Indeed, in any aspect of the invention, the CRISPR complex may comprisea CRISPR effector protein complexed with a guide sequence hybridized orhybridizable to a target sequence. Similar considerations and conditionsapply as above for methods of modifying a target polynucleotide.

Thus in any of the non-naturally-occurring CRISPR effector proteinsdescribed herein comprise at least one modification and whereby theeffector protein has certain improved capabilities. In particular, anyof the effector proteins are capable of forming a CRISPR complex with aguide RNA. When such a complex forms, the guide RNA is capable ofbinding to a target polynucleotide sequence and the effector protein iscapable of modifying a target locus. In addition, the effector proteinin the CRISPR complex has reduced capability of modifying one or moreoff-target loci as compared to an unmodified enzyme/effector protein.

In addition, the modified CRISPR enzymes described herein encompassenzymes whereby in the CRISPR complex the effector protein has increasedcapability of modifying the one or more target loci as compared to anunmodified enzyme/effector protein. Such function may be providedseparate to or provided in combination with the above-described functionof reduced capability of modifying one or more off-target loci. Any sucheffector proteins may be provided with any of the further modificationsto the CRISPR effector protein as described herein, such as incombination with any activity provided by one or more associatedheterologous functional domains, any further mutations to reducenuclease activity and the like.

In advantageous embodiments of the invention, the modified CRISPReffector protein is provided with reduced capability of modifying one ormore off-target loci as compared to an unmodified enzyme/effectorprotein and increased capability of modifying the one or more targetloci as compared to an unmodified enzyme/effector protein. Incombination with further modifications to the effector protein,significantly enhanced specificity may be achieved. For example,combination of such advantageous embodiments with one or more additionalmutations is provided wherein the one or more additional mutations arein one or more catalytically active domains. Such further catalyticmutations may confer nickase functionality as described in detailelsewhere herein. In such effector proteins, enhanced specificity may beachieved due to an improved specificity in terms of effector proteinactivity.

Modifications to reduce off-target effects and/or enhance on-targeteffects as described above may be made to amino acid residues located ina positively-charged region/groove situated between the RuvC-III and HNHdomains. It will be appreciated that any of the functional effectsdescribed above may be achieved by modification of amino acids withinthe aforementioned groove but also by modification of amino acidsadjacent to or outside of that groove.

Additional functionalities which may be engineered into modified CRISPReffector proteins as described herein include the following. 1. modifiedCRISPR effector proteins that disrupt DNA:protein interactions withoutaffecting protein tertiary or secondary structure. This includesresidues that contact any part of the RNA:DNA duplex. 2. modified CRISPReffector proteins that weaken intra-protein interactions holding C2c1 orC2c3 in conformation essential for nuclease cutting in response to DNAbinding (on or off target). For example: a modification that mildlyinhibits, but still allows, the nuclease conformation of the HNH domain(positioned at the scissile phosphate). 3. modified CRISPR effectorproteins that strengthen intra-protein interactions holding C2c1 or C2c3in a conformation inhibiting nuclease activity in response to DNAbinding (on or off targets). For example: a modification that stabilizesthe HNH domain in a conformation away from the scissile phosphate. Anysuch additional functional enhancement may be provided in combinationwith any other modification to the CRISPR effector protein as describedin detail elsewhere herein.

Any of the herein described improved functionalities may be made to anyCRISPR effector protein, such as a C2c1 or C2c3 effector protein.However, it will be appreciated that any of the functionalitiesdescribed herein may be engineered into C2c1 or C2c3 effector proteinsfrom other orthologs, including chimeric effector proteins comprisingfragments from multiple orthologs.

Nucleic Acids, Amino Acids and Proteins, Regulatory Sequences, Vectors,Etc.

The invention uses nucleic acids to bind target DNA sequences. This isadvantageous as nucleic acids are much easier and cheaper to producethan proteins, and the specificity can be varied according to the lengthof the stretch where homology is sought. Complex 3-D positioning ofmultiple fingers, for example is not required. The terms“polynucleotide”, “nucleotide”, “nucleotide sequence”, “nucleic acid”and “oligonucleotide” are used interchangeably. They refer to apolymeric form of nucleotides of any length, either deoxyribonucleotidesor ribonucleotides, or analogs thereof. Polynucleotides may have anythree dimensional structure, and may perform any function, known orunknown. The following are non-limiting examples of polynucleotides:coding or non-coding regions of a gene or gene fragment, loci (locus)defined from linkage analysis, exons, introns, messenger RNA (mRNA),transfer RNA, ribosomal RNA, short interfering RNA (siRNA),short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. The term also encompassesnucleic-acid-like structures with synthetic backbones, see, e.g.,Eckstein, 1991; Baserga et al., 1992; Milligan, 1993; WO 97/03211; WO96/39154; Mata, 1997; Strauss-Soukup, 1997; and Samstag, 1996. Apolynucleotide may comprise one or more modified nucleotides, such asmethylated nucleotides and nucleotide analogs. If present, modificationsto the nucleotide structure may be imparted before or after assembly ofthe polymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization, such as by conjugation with a labeling component.As used herein the term “wild type” is a term of the art understood byskilled persons and means the typical form of an organism, strain, geneor characteristic as it occurs in nature as distinguished from mutant orvariant forms. A “wild type” can be a base line. As used herein the term“variant” should be taken to mean the exhibition of qualities that havea pattern that deviates from what occurs in nature. The terms“non-naturally occurring” or “engineered” are used interchangeably andindicate the involvement of the hand of man. The terms, when referringto nucleic acid molecules or polypeptides mean that the nucleic acidmolecule or the polypeptide is at least substantially free from at leastone other component with which they are naturally associated in natureand as found in nature. “Complementarity” refers to the ability of anucleic acid to form hydrogen bond(s) with another nucleic acid sequenceby either traditional Watson-Crick base pairing or other non-traditionaltypes. A percent complementarity indicates the percentage of residues ina nucleic acid molecule which can form hydrogen bonds (e.g.,Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5,6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100%complementary). “Perfectly complementary” means that all the contiguousresidues of a nucleic acid sequence will hydrogen bond with the samenumber of contiguous residues in a second nucleic acid sequence.“Substantially complementary” as used herein refers to a degree ofcomplementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or morenucleotides, or refers to two nucleic acids that hybridize understringent conditions. As used herein, “stringent conditions” forhybridization refer to conditions under which a nucleic acid havingcomplementarity to a target sequence predominantly hybridizes with thetarget sequence, and substantially does not hybridize to non-targetsequences. Stringent conditions are generally sequence-dependent, andvary depending on a number of factors. In general, the longer thesequence, the higher the temperature at which the sequence specificallyhybridizes to its target sequence. Non-limiting examples of stringentconditions are described in detail in Tijssen (1993), LaboratoryTechniques In Biochemistry And Molecular Biology-Hybridization WithNucleic Acid Probes Part I, Second Chapter “Overview of principles ofhybridization and the strategy of nucleic acid probe assay”, Elsevier,N.Y. Where reference is made to a polynucleotide sequence, thencomplementary or partially complementary sequences are also envisaged.These are preferably capable of hybridizing to the reference sequenceunder highly stringent conditions. Generally, in order to maximize thehybridization rate, relatively low-stringency hybridization conditionsare selected: about 20 to 250 C lower than the thermal melting point(T_(m)). The T_(m) is the temperature at which 50% of specific targetsequence hybridizes to a perfectly complementary probe in solution at adefined ionic strength and pH. Generally, in order to require at leastabout 85% nucleotide complementarity of hybridized sequences, highlystringent washing conditions are selected to be about 5 to 150 C lowerthan the T_(m). In order to require at least about 70% nucleotidecomplementarity of hybridized sequences, moderately-stringent washingconditions are selected to be about 15 to 300 C lower than the T_(m).Highly permissive (very low stringency) washing conditions may be as lowas 50° C. below the T_(m), allowing a high level of mis-matching betweenhybridized sequences. Those skilled in the art will recognize that otherphysical and chemical parameters in the hybridization and wash stagescan also be altered to affect the outcome of a detectable hybridizationsignal from a specific level of homology between target and probesequences. Preferred highly stringent conditions comprise incubation in50% formamide, 5×SSC, and 1% SDS at 42° C., or incubation in 5×SSC and1% SDS at 65° C., with wash in 0.2×SSC and 0.1% SDS at 65° C.“Hybridization” refers to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson Crick base pairing, Hoogstein binding, or inany other sequence specific manner. The complex may comprise two strandsforming a duplex structure, three or more strands forming a multistranded complex, a single self-hybridizing strand, or any combinationof these. A hybridization reaction may constitute a step in a moreextensive process, such as the initiation of PCR, or the cleavage of apolynucleotide by an enzyme. A sequence capable of hybridizing with agiven sequence is referred to as the “complement” of the given sequence.As used herein, the term “genomic locus” or “locus” (plural loci) is thespecific location of a gene or DNA sequence on a chromosome. A “gene”refers to stretches of DNA or RNA that encode a polypeptide or an RNAchain that has functional role to play in an organism and hence is themolecular unit of heredity in living organisms. For the purpose of thisinvention it may be considered that genes include regions which regulatethe production of the gene product, whether or not such regulatorysequences are adjacent to coding and/or transcribed sequences.Accordingly, a gene includes, but is not necessarily limited to,promoter sequences, terminators, translational regulatory sequences suchas ribosome binding sites and internal ribosome entry sites, enhancers,silencers, insulators, boundary elements, replication origins, matrixattachment sites and locus control regions. As used herein, “expressionof a genomic locus” or “gene expression” is the process by whichinformation from a gene is used in the synthesis of a functional geneproduct. The products of gene expression are often proteins, but innon-protein coding genes such as rRNA genes or tRNA genes, the productis functional RNA. The process of gene expression is used by all knownlife-eukaryotes (including multicellular organisms), prokaryotes(bacteria and archaea) and viruses to generate functional products tosurvive. As used herein “expression” of a gene or nucleic acidencompasses not only cellular gene expression, but also thetranscription and translation of nucleic acid(s) in cloning systems andin any other context. As used herein, “expression” also refers to theprocess by which a polynucleotide is transcribed from a DNA template(such as into and mRNA or other RNA transcript) and/or the process bywhich a transcribed mRNA is subsequently translated into peptides,polypeptides, or proteins. Transcripts and encoded polypeptides may becollectively referred to as “gene product.” If the polynucleotide isderived from genomic DNA, expression may include splicing of the mRNA ina eukaryotic cell. The terms “polypeptide”, “peptide” and “protein” areused interchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation, such asconjugation with a labeling component. As used herein the term “aminoacid” includes natural and/or unnatural or synthetic amino acids,including glycine and both the D or L optical isomers, and amino acidanalogs and peptidomimetics. As used herein, the term “domain” or“protein domain” refers to a part of a protein sequence that may existand function independently of the rest of the protein chain. Asdescribed in aspects of the invention, sequence identity is related tosequence homology. Homology comparisons may be conducted by eye, or moreusually, with the aid of readily available sequence comparison programs.These commercially available computer programs may calculate percent (%)homology between two or more sequences and may also calculate thesequence identity shared by two or more amino acid or nucleic acidsequences.

In aspects of the invention the term “guide RNA”, refers to thepolynucleotide sequence comprising one or more of a putative oridentified tracr sequence and a putative or identified crRNA sequence orguide sequence. In particular embodiments, the “guide RNA” comprises aputative or identified crRNA sequence or guide sequence. In furtherembodiments, the guide RNA does not comprise a putative or identifiedtracr sequence.

As used herein the term “wild type” is a term of the art understood byskilled persons and means the typical form of an organism, strain, geneor characteristic as it occurs in nature as distinguished from mutant orvariant forms. A “wild type” can be a base line.

As used herein the term “variant” should be taken to mean the exhibitionof qualities that have a pattern that deviates from what occurs innature.

The terms “non-naturally occurring” or “engineered” are usedinterchangeably and indicate the involvement of the hand of man. Theterms, when referring to nucleic acid molecules or polypeptides meanthat the nucleic acid molecule or the polypeptide is at leastsubstantially free from at least one other component with which they arenaturally associated in nature and as found in nature. In all aspectsand embodiments, whether they include these terms or not, it will beunderstood that, preferably, the may be optional and thus preferablyincluded or not preferably not included. Furthermore, the terms“non-naturally occurring” and “engineered” may be used interchangeablyand so can therefore be used alone or in combination and one or othermay replace mention of both together. In particular, “engineered” ispreferred in place of “non-naturally occurring” or “non-naturallyoccurring and/or engineered.”

Sequence homologies may be generated by any of a number of computerprograms known in the art, for example BLAST or FASTA, etc. A suitablecomputer program for carrying out such an alignment is the GCG WisconsinBestfit package (University of Wisconsin, U.S.A; Devereux et al., 1984,Nucleic Acids Research 12:387). Examples of other software than mayperform sequence comparisons include, but are not limited to, the BLASTpackage (see Ausubel et al., 1999 ibid—Chapter 18), FASTA (Atschul etal., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparisontools. Both BLAST and FASTA are available for offline and onlinesearching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60). Howeverit is preferred to use the GCG Bestfit program. Percentage (%) sequencehomology may be calculated over contiguous sequences, i.e., one sequenceis aligned with the other sequence and each amino acid or nucleotide inone sequence is directly compared with the corresponding amino acid ornucleotide in the other sequence, one residue at a time. This is calledan “ungapped” alignment. Typically, such ungapped alignments areperformed only over a relatively short number of residues. Although thisis a very simple and consistent method, it fails to take intoconsideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion may cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without unduly penalizing the overall homology or identityscore. This is achieved by inserting “gaps” in the sequence alignment totry to maximize local homology or identity. However, these more complexmethods assign “gap penalties” to each gap that occurs in the alignmentso that, for the same number of identical amino acids, a sequencealignment with as few gaps as possible—reflecting higher relatednessbetween the two compared sequences—may achieve a higher score than onewith many gaps. “Affinity gap costs” are typically used that charge arelatively high cost for the existence of a gap and a smaller penaltyfor each subsequent residue in the gap. This is the most commonly usedgap scoring system. High gap penalties may, of course, produce optimizedalignments with fewer gaps. Most alignment programs allow the gappenalties to be modified. However, it is preferred to use the defaultvalues when using such software for sequence comparisons. For example,when using the GCG Wisconsin Bestfit package the default gap penalty foramino acid sequences is −12 for a gap and −4 for each extension.Calculation of maximum % homology therefore first requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (Devereux et al., 1984Nuc. Acids Research 12 p387). Examples of other software than mayperform sequence comparisons include, but are not limited to, the BLASTpackage (see Ausubel et al., 1999 Short Protocols in Molecular Biology,4^(th) Ed.—Chapter 18), FASTA (Altschul et al., 1990 J. Mol. Biol.403-410) and the GENEWORKS suite of comparison tools. Both BLAST andFASTA are available for offline and online searching (see Ausubel etal., 1999, Short Protocols in Molecular Biology, pages 7-58 to 7-60).However, for some applications, it is preferred to use the GCG Bestfitprogram. A new tool, called BLAST 2 Sequences is also available forcomparing protein and nucleotide sequences (see FEMS Microbiol Lett.1999 174(2): 247-50; FEMS Microbiol Lett. 1999 177(1): 187-8 and thewebsite of the National Center for Biotechnology information at thewebsite of the National Institutes for Health). Although the final %homology may be measured in terms of identity, the alignment processitself is typically not based on an all-or-nothing pair comparison.Instead, a scaled similarity score matrix is generally used that assignsscores to each pair-wise comparison based on chemical similarity orevolutionary distance. An example of such a matrix commonly used is theBLOSUM62 matrix—the default matrix for the BLAST suite of programs. GCGWisconsin programs generally use either the public default values or acustom symbol comparison table, if supplied (see user manual for furtherdetails). For some applications, it is preferred to use the publicdefault values for the GCG package, or in the case of other software,the default matrix, such as BLOSUM62. Alternatively, percentagehomologies may be calculated using the multiple alignment feature inDNASIS™ (Hitachi Software), based on an algorithm, analogous to CLUSTAL(Higgins D G & Sharp P M (1988), Gene 73(1), 237-244). Once the softwarehas produced an optimal alignment, it is possible to calculate %homology, preferably % sequence identity. The software typically doesthis as part of the sequence comparison and generates a numericalresult. The sequences may also have deletions, insertions orsubstitutions of amino acid residues which produce a silent change andresult in a functionally equivalent substance. Deliberate amino acidsubstitutions may be made on the basis of similarity in amino acidproperties (such as polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues) and it istherefore useful to group amino acids together in functional groups.Amino acids may be grouped together based on the properties of theirside chains alone. However, it is more useful to include mutation dataas well. The sets of amino acids thus derived are likely to be conservedfor structural reasons. These sets may be described in the form of aVenn diagram (Livingstone C. D. and Barton G. J. (1993) “Proteinsequence alignments: a strategy for the hierarchical analysis of residueconservation” Comput. Appl. Biosci. 9: 745-756) (Taylor W. R. (1986)“The classification of amino acid conservation” J. Theor. Biol. 119;205-218). Conservative substitutions may be made, for example accordingto the table below which describes a generally accepted Venn diagramgrouping of amino acids.

TABLE 2 Set Sub-set Hydrophobic F W Y H K M I L V A G C Aromatic F W Y HAliphatic I L V Polar W Y H K R E D C S T N Q Charged H K R E DPositively H K R charged Negatively E D charged Small V C A G S P T N DTiny A G S

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein to refer to a vertebrate, preferably a mammal,more preferably a human. Mammals include, but are not limited to,murines, simians, humans, farm animals, sport animals, and pets.Tissues, cells and their progeny of a biological entity obtained in vivoor cultured in vitro are also encompassed.

The terms “therapeutic agent”, “therapeutic capable agent” or “treatmentagent” are used interchangeably and refer to a molecule or compound thatconfers some beneficial effect upon administration to a subject. Thebeneficial effect includes enablement of diagnostic determinations;amelioration of a disease, symptom, disorder, or pathological condition;reducing or preventing the onset of a disease, symptom, disorder orcondition; and generally counteracting a disease, symptom, disorder orpathological condition.

As used herein, “treatment” or “treating,” or “palliating” or“ameliorating” are used interchangeably. These terms refer to anapproach for obtaining beneficial or desired results including but notlimited to a therapeutic benefit and/or a prophylactic benefit. Bytherapeutic benefit is meant any therapeutically relevant improvement inor effect on one or more diseases, conditions, or symptoms undertreatment. For prophylactic benefit, the compositions may beadministered to a subject at risk of developing a particular disease,condition, or symptom, or to a subject reporting one or more of thephysiological symptoms of a disease, even though the disease, condition,or symptom may not have yet been manifested.

The term “effective amount” or “therapeutically effective amount” refersto the amount of an agent that is sufficient to effect beneficial ordesired results. The therapeutically effective amount may vary dependingupon one or more of: the subject and disease condition being treated,the weight and age of the subject, the severity of the diseasecondition, the manner of administration and the like, which can readilybe determined by one of ordinary skill in the art. The term also appliesto a dose that will provide an image for detection by any one of theimaging methods described herein. The specific dose may vary dependingon one or more of: the particular agent chosen, the dosing regimen to befollowed, whether it is administered in combination with othercompounds, timing of administration, the tissue to be imaged, and thephysical delivery system in which it is carried.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of immunology, biochemistry,chemistry, molecular biology, microbiology, cell biology, genomics andrecombinant DNA, which are within the skill of the art. See Sambrook,Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2ndedition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel,et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press,Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, ALABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)).

Several aspects of the invention relate to vector systems comprising oneor more vectors, or vectors as such. Vectors can be designed forexpression of CRISPR transcripts (e.g. nucleic acid transcripts,proteins, or enzymes) in prokaryotic or eukaryotic cells. For example,CRISPR transcripts can be expressed in bacterial cells such asEscherichia coli, insect cells (using baculovirus expression vectors),yeast cells, or mammalian cells. Suitable host cells are discussedfurther in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY185, Academic Press, San Diego, Calif. (1990). Alternatively, therecombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Embodiments of the invention include sequences (both polynucleotide orpolypeptide) which may comprise homologous substitution (substitutionand replacement are both used herein to mean the interchange of anexisting amino acid residue or nucleotide, with an alternative residueor nucleotide) that may occur i.e., like-for-like substitution in thecase of amino acids such as basic for basic, acidic for acidic, polarfor polar, etc. Non-homologous substitution may also occur i.e., fromone class of residue to another or alternatively involving the inclusionof unnatural amino acids such as ornithine (hereinafter referred to asZ), diaminobutyric acid ornithine (hereinafter referred to as B),norleucine ornithine (hereinafter referred to as O), pyridylalanine,thienylalanine, naphthylalanine and phenylglycine. Variant amino acidsequences may include suitable spacer groups that may be insertedbetween any two amino acid residues of the sequence including alkylgroups such as methyl, ethyl or propyl groups in addition to amino acidspacers such as glycine or β-alanine residues. A further form ofvariation, which involves the presence of one or more amino acidresidues in peptoid form, may be well understood by those skilled in theart. For the avoidance of doubt, “the peptoid form” is used to refer tovariant amino acid residues wherein the α-carbon substituent group is onthe residue's nitrogen atom rather than the α-carbon. Processes forpreparing peptides in the peptoid form are known in the art, for exampleSimon R J et al., PNAS (1992) 89(20), 9367-9371 and Horwell D C, TrendsBiotechnol. (1995) 13(4), 132-134.

Homology modelling: Corresponding residues in other C2c1 or C2c3orthologs can be identified by the methods of Zhang et al., 2012(Nature; 490(7421): 556-60) and Chen et al., 2015 (PLoS Comput Biol;11(5): e1004248)—a computational protein-protein interaction (PPI)method to predict interactions mediated by domain-motif interfaces.PrePPI (Predicting PPI), a structure based PPI prediction method,combines structural evidence with non-structural evidence using aBayesian statistical framework. The method involves taking a pair aquery proteins and using structural alignment to identify structuralrepresentatives that correspond to either their experimentallydetermined structures or homology models. Structural alignment isfurther used to identify both close and remote structural neighbors byconsidering global and local geometric relationships. Whenever twoneighbors of the structural representatives form a complex reported inthe Protein Data Bank, this defines a template for modelling theinteraction between the two query proteins. Models of the complex arecreated by superimposing the representative structures on theircorresponding structural neighbor in the template. This approach isfurther described in Dey et al., 2013 (Prot Sci; 22: 359-66).

For purpose of this invention, amplification means any method employinga primer and a polymerase capable of replicating a target sequence withreasonable fidelity. Amplification may be carried out by natural orrecombinant DNA polymerases such as TaqGold™, T7 DNA polymerase, Klenowfragment of E. coli DNA polymerase, and reverse transcriptase. Apreferred amplification method is PCR.

In certain aspects the invention involves vectors. A used herein, a“vector” is a tool that allows or facilitates the transfer of an entityfrom one environment to another. It is a replicon, such as a plasmid,phage, or cosmid, into which another DNA segment may be inserted so asto bring about the replication of the inserted segment. Generally, avector is capable of replication when associated with the proper controlelements. In general, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. Vectors include, but are not limited to, nucleic acidmolecules that are single-stranded, double-stranded, or partiallydouble-stranded; nucleic acid molecules that comprise one or more freeends, no free ends (e.g., circular); nucleic acid molecules thatcomprise DNA, RNA, or both; and other varieties of polynucleotides knownin the art. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments canbe inserted, such as by standard molecular cloning techniques. Anothertype of vector is a viral vector, wherein virally-derived DNA or RNAsequences are present in the vector for packaging into a virus (e.g.,retroviruses, replication defective retroviruses, adenoviruses,replication defective adenoviruses, and adeno-associated viruses(AAVs)). Viral vectors also include polynucleotides carried by a virusfor transfection into a host cell. Certain vectors are capable ofautonomous replication in a host cell into which they are introduced(e.g., bacterial vectors having a bacterial origin of replication andepisomal mammalian vectors). Other vectors (e.g., non-episomal mammalianvectors) are integrated into the genome of a host cell upon introductioninto the host cell, and thereby are replicated along with the hostgenome. Moreover, certain vectors are capable of directing theexpression of genes to which they are operatively-linked. Such vectorsare referred to herein as “expression vectors.” Common expressionvectors of utility in recombinant DNA techniques are often in the formof plasmids.

Recombinant expression vectors can comprise a nucleic acid of theinvention in a form suitable for expression of the nucleic acid in ahost cell, which means that the recombinant expression vectors includeone or more regulatory elements, which may be selected on the basis ofthe host cells to be used for expression, that is operatively-linked tothe nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory element(s)in a manner that allows for expression of the nucleotide sequence (e.g.,in an in vitro transcription/translation system or in a host cell whenthe vector is introduced into the host cell). With regards torecombination and cloning methods, mention is made of U.S. patentapplication Ser. No. 10/815,730, published Sep. 2, 2004 as US2004-0171156 A1, the contents of which are herein incorporated byreference in their entirety.

Aspects of the invention relate to bicistronic vectors for guide RNA andwild type, modified or mutated CRISPR effector proteins/enzymes (e.g.C2c1 or C2c3). Bicistronic expression vectors guide RNA and wild type,modified or mutated CRISPR effector proteins/enzymes (e.g. C2c1 or C2c3)are preferred. In general and particularly in this embodiment and wildtype, modified or mutated CRISPR effector proteins/enzymes (e.g. C2c1 orC2c3) is preferably driven by the CBh promoter. The RNA may preferablybe driven by a Pol III promoter, such as a U6 promoter. Ideally the twoare combined.

In some embodiments, a loop in the guide RNA is provided. This may be astem loop or a tetra loop. The loop is preferably GAAA, but it is notlimited to this sequence or indeed to being only 4 bp in length. Indeed,preferred loop forming sequences for use in hairpin structures are fournucleotides in length, and most preferably have the sequence GAAA.However, longer or shorter loop sequences may be used, as mayalternative sequences. The sequences preferably include a nucleotidetriplet (for example, AAA), and an additional nucleotide (for example Cor G). Examples of loop forming sequences include CAAA and AAAG.

In practicing any of the methods disclosed herein, a suitable vector canbe introduced to a cell or an embryo via one or more methods known inthe art, including without limitation, microinjection, electroporation,sonoporation, biolistics, calcium phosphate-mediated transfection,cationic transfection, liposome transfection, dendrimer transfection,heat shock transfection, nucleofection transfection, magnetofection,lipofection, impalefection, optical transfection, proprietaryagent-enhanced uptake of nucleic acids, and delivery via liposomes,immunoliposomes, virosomes, or artificial virions. In some methods, thevector is introduced into an embryo by microinjection. The vector orvectors may be microinjected into the nucleus or the cytoplasm of theembryo. In some methods, the vector or vectors may be introduced into acell by nucleofection.

The term “regulatory element” is intended to include promoters,enhancers, internal ribosomal entry sites (IRES), and other expressioncontrol elements (e.g., transcription termination signals, such aspolyadenylation signals and poly-U sequences). Such regulatory elementsare described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).Regulatory elements include those that direct constitutive expression ofa nucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). A tissue-specific promoter maydirect expression primarily in a desired tissue of interest, such asmuscle, neuron, bone, skin, blood, specific organs (e.g., liver,pancreas), or particular cell types (e.g., lymphocytes). Regulatoryelements may also direct expression in a temporal-dependent manner, suchas in a cell-cycle dependent or developmental stage-dependent manner,which may or may not also be tissue or cell-type specific. In someembodiments, a vector comprises one or more pol III promoter (e.g., 1,2, 3, 4, 5, or more pol III promoters), one or more pol II promoters(e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol Ipromoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), orcombinations thereof. Examples of pol III promoters include, but are notlimited to, U6 and H1 promoters. Examples of pol II promoters include,but are not limited to, the retroviral Rous sarcoma virus (RSV) LTRpromoter (optionally with the RSV enhancer), the cytomegalovirus (CMV)promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al,Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductasepromoter, the β-actin promoter, the phosphoglycerol kinase (PGK)promoter, and the EF1α promoter. Also encompassed by the term“regulatory element” are enhancer elements, such as WPRE; CMV enhancers;the R-U5′ segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p.466-472, 1988); SV40 enhancer; and the intron sequence between exons 2and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p.1527-31, 1981). It will be appreciated by those skilled in the art thatthe design of the expression vector can depend on such factors as thechoice of the host cell to be transformed, the level of expressiondesired, etc. A vector can be introduced into host cells to therebyproduce transcripts, proteins, or peptides, including fusion proteins orpeptides, encoded by nucleic acids as described herein (e.g., clusteredregularly interspersed short palindromic repeats (CRISPR) transcripts,proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.).With regards to regulatory sequences, mention is made of U.S. patentapplication Ser. No. 10/491,026, the contents of which are incorporatedby reference herein in their entirety. With regards to promoters,mention is made of PCT publication WO 2011/028929 and U.S. applicationSer. No. 12/511,940, the contents of which are incorporated by referenceherein in their entirety.

Vectors can be designed for expression of CRISPR transcripts (e.g.,nucleic acid transcripts, proteins, or enzymes) in prokaryotic oreukaryotic cells. For example, CRISPR transcripts can be expressed inbacterial cells such as Escherichia coli, insect cells (usingbaculovirus expression vectors), yeast cells, or mammalian cells.Suitable host cells are discussed further in Goeddel, GENE EXPRESSIONTECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.(1990). Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

Vectors may be introduced and propagated in a prokaryote or prokaryoticcell. In some embodiments, a prokaryote is used to amplify copies of avector to be introduced into a eukaryotic cell or as an intermediatevector in the production of a vector to be introduced into a eukaryoticcell (e.g., amplifying a plasmid as part of a viral vector packagingsystem). In some embodiments, a prokaryote is used to amplify copies ofa vector and express one or more nucleic acids, such as to provide asource of one or more proteins for delivery to a host cell or hostorganism. Expression of proteins in prokaryotes is most often carriedout in Escherichia coli with vectors containing constitutive orinducible promoters directing the expression of either fusion ornon-fusion proteins. Fusion vectors add a number of amino acids to aprotein encoded therein, such as to the amino terminus of therecombinant protein. Such fusion vectors may serve one or more purposes,such as: (i) to increase expression of recombinant protein; (ii) toincrease the solubility of the recombinant protein; and (iii) to aid inthe purification of the recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase. Example fusionexpression vectors include pGEX (Pharmacia Biotech Inc; Smith andJohnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly,Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d(Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185,Academic Press, San Diego, Calif. (1990) 60-89).

In some embodiments, a vector is a yeast expression vector. Examples ofvectors for expression in yeast Saccharomyces cerevisae include pYepSec1(Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kuijan andHerskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), andpicZ (InVitrogen Corp, San Diego, Calif.).

In some embodiments, a vector drives protein expression in insect cellsusing baculovirus expression vectors. Baculovirus vectors available forexpression of proteins in cultured insect cells (e.g., SF9 cells)include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170:31-39).

In some embodiments, a vector is capable of driving expression of one ormore sequences in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, 1987.Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195).When used in mammalian cells, the expression vector's control functionsare typically provided by one or more regulatory elements. For example,commonly used promoters are derived from polyoma, adenovirus 2,cytomegalovirus, simian virus 40, and others disclosed herein and knownin the art. For other suitable expression systems for both prokaryoticand eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al.,MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989.

In some embodiments, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert, et al.,1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame andEaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of Tcell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) andimmunoglobulins (Baneiji, et al., 1983. Cell 33: 729-740; Queen andBaltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci.USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985.Science 230: 912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990.Science 249: 374-379) and the α-fetoprotein promoter (Campes andTilghman, 1989. Genes Dev. 3: 537-546). With regards to theseprokaryotic and eukaryotic vectors, mention is made of U.S. Pat. No.6,750,059, the contents of which are incorporated by reference herein intheir entirety. Other embodiments of the invention may relate to the useof viral vectors, with regards to which mention is made of U.S. patentapplication Ser. No. 13/092,085, the contents of which are incorporatedby reference herein in their entirety. Tissue-specific regulatoryelements are known in the art and in this regard, mention is made ofU.S. Pat. No. 7,776,321, the contents of which are incorporated byreference herein in their entirety.

In some embodiments, a regulatory element is operably linked to one ormore elements of a CRISPR system so as to drive expression of the one ormore elements of the CRISPR system. In general, CRISPRs (ClusteredRegularly Interspaced Short Palindromic Repeats), also known as SPIDRs(SPacer Interspersed Direct Repeats), constitute a family of DNA locithat are usually specific to a particular bacterial species. The CRISPRlocus comprises a distinct class of interspersed short sequence repeats(SSRs) that were recognized in E. coli (Ishino et al., J. Bacteriol.,169:5429-5433 [1987]; and Nakata et al., J. Bacteriol., 171:3553-3556[1989]), and associated genes. Similar interspersed SSRs have beenidentified in Haloferax mediterranei, Streptococcus pyogenes, Anabaena,and Mycobacterium tuberculosis (See, Groenen et al., Mol. Microbiol.,10:1057-1065 [1993]; Hoe et al., Emerg. Infect. Dis., 5:254-263 [1999];Masepohl et al., Biochim. Biophys. Acta 1307:26-30 [1996]; and Mojica etal., Mol. Microbiol., 17:85-93 [1995]). The CRISPR loci typically differfrom other SSRs by the structure of the repeats, which have been termedshort regularly spaced repeats (SRSRs) (Janssen et al., OMICS J. Integ.Biol., 6:23-33 [2002]; and Mojica et al., Mol. Microbiol., 36:244-246[2000]). In general, the repeats are short elements that occur inclusters that are regularly spaced by unique intervening sequences witha substantially constant length (Mojica et al., [2000], supra). Althoughthe repeat sequences are highly conserved between strains, the number ofinterspersed repeats and the sequences of the spacer regions typicallydiffer from strain to strain (van Embden et al., J. Bacteriol.,182:2393-2401 [2000]). CRISPR loci have been identified in more than 40prokaryotes (See e.g., Jansen et al., Mol. Microbiol., 43:1565-1575[2002]; and Mojica et al., [2005]) including, but not limited toAeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Haloarcula,Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus,Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium, Mycobacterium,Streptomyces, Aquifex, Porphyromonas, Chlorobium, Thermus, Bacillus,Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma,Fusobacterium, Azoarcus, Chromobacterium, Neisseria, Nitrosomonas,Desulfovibrio, Geobacter, Myxococcus, Campylobacter, Wolinella,Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus,Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia,Treponema, and Thermotoga.

In general, “nucleic acid-targeting system” as used in the presentapplication refers collectively to transcripts and other elementsinvolved in the expression of or directing the activity of nucleicacid-targeting CRISPR-associated (“Cas”) genes (also referred to hereinas an effector protein), including sequences encoding a nucleicacid-targeting Cas (effector) protein and a guide RNA (comprising crRNAsequence and a trans-activating CRISPR/Cas system RNA (tracrRNA)sequence), or other sequences and transcripts from a nucleicacid-targeting CRISPR locus. In some embodiments, one or more elementsof a nucleic acid-targeting system are derived from a Type V/Type VInucleic acid-targeting CRISPR system. In some embodiments, one or moreelements of a nucleic acid-targeting system is derived from a particularorganism comprising an endogenous nucleic acid-targeting CRISPR system.In general, a nucleic acid-targeting system is characterized by elementsthat promote the formation of a nucleic acid-targeting complex at thesite of a target sequence. In the context of formation of a nucleicacid-targeting complex, “target sequence” refers to a sequence to whicha guide sequence is designed to have complementarity, wherehybridization between a target sequence and a guide RNA promotes theformation of a DNA or RNA-targeting complex. Full complementarity is notnecessarily required, provided there is sufficient complementarity tocause hybridization and promote formation of a nucleic acid-targetingcomplex. A target sequence may comprise RNA polynucleotides. In someembodiments, a target sequence is located in the nucleus or cytoplasm ofa cell. In some embodiments, the target sequence may be within anorganelle of a eukaryotic cell, for example, mitochondrion orchloroplast. A sequence or template that may be used for recombinationinto the targeted locus comprising the target sequences is referred toas an “editing template” or “editing RNA” or “editing sequence”. Inaspects of the invention, an exogenous template RNA may be referred toas an editing template. In an aspect of the invention the recombinationis homologous recombination.

Typically, in the context of an endogenous nucleic acid-targetingsystem, formation of a nucleic acid-targeting complex (comprising aguide RNA hybridized to a target sequence and complexed with one or morenucleic acid-targeting effector proteins) results in cleavage of one orboth RNA strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 50, or more base pairs from) the target sequence. In someembodiments, one or more vectors driving expression of one or moreelements of a nucleic acid-targeting system are introduced into a hostcell such that expression of the elements of the nucleic acid-targetingsystem direct formation of a nucleic acid-targeting complex at one ormore target sites. For example, a nucleic acid-targeting effectorprotein and a guide RNA could each be operably linked to separateregulatory elements on separate vectors. Alternatively, two or more ofthe elements expressed from the same or different regulatory elements,may be combined in a single vector, with one or more additional vectorsproviding any components of the nucleic acid-targeting system notincluded in the first vector. nucleic acid-targeting system elementsthat are combined in a single vector may be arranged in any suitableorientation, such as one element located 5′ with respect to (“upstream”of) or 3′ with respect to (“downstream” of) a second element. The codingsequence of one element may be located on the same or opposite strand ofthe coding sequence of a second element, and oriented in the same oropposite direction. In some embodiments, a single promoter drivesexpression of a transcript encoding a nucleic acid-targeting effectorprotein and a guide RNA embedded within one or more intron sequences(e.g. each in a different intron, two or more in at least one intron, orall in a single intron). In some embodiments, the nucleic acid-targetingeffector protein and guide RNA are operably linked to and expressed fromthe same promoter.

In general, a guide sequence is any polynucleotide sequence havingsufficient complementarity with a target polynucleotide sequence tohybridize with the target sequence and direct sequence-specific bindingof a nucleic acid-targeting complex to the target sequence. In someembodiments, the degree of complementarity between a guide sequence andits corresponding target sequence, when optimally aligned using asuitable alignment algorithm, is about or more than about 50%, 60%, 75%,80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may bedetermined with the use of any suitable algorithm for aligningsequences, non-limiting example of which include the Smith-Watermanalgorithm, the Needleman-Wunsch algorithm, algorithms based on theBurrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW,Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, SanDiego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq(available at maq.sourceforge.net). In some embodiments, a guidesequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75,or more nucleotides in length. In some embodiments, a guide sequence isless than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewernucleotides in length. The ability of a guide sequence to directsequence-specific binding of a nucleic acid-targeting complex to atarget sequence may be assessed by any suitable assay. For example, thecomponents of a nucleic acid-targeting system sufficient to form anucleic acid-targeting complex, including the guide sequence to betested, may be provided to a host cell having the corresponding targetsequence, such as by transfection with vectors encoding the componentsof the nucleic acid-targeting CRISPR sequence, followed by an assessmentof preferential cleavage within or in the vicinity of the targetsequence, such as by Surveyor assay as described herein. Similarly,cleavage of a target polynucleotide sequence (or a sequence in thevicinity thereof) may be evaluated in a test tube by providing thetarget sequence, components of a nucleic acid-targeting complex,including the guide sequence to be tested and a control guide sequencedifferent from the test guide sequence, and comparing binding or rate ofcleavage at or in the vicinity of the target sequence between the testand control guide sequence reactions. Other assays are possible, andwill occur to those skilled in the art.

A guide sequence may be selected to target any target sequence. In someembodiments, the target sequence is a sequence within a gene transcriptor mRNA.

In some embodiments, the target sequence is a sequence within a genomeof a cell.

In some embodiments, a guide sequence is selected to reduce the degreeof secondary structure within the guide sequence. Secondary structuremay be determined by any suitable polynucleotide folding algorithm. Someprograms are based on calculating the minimal Gibbs free energy. Anexample of one such algorithm is mFold, as described by Zuker andStiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example foldingalgorithm is the online webserver RNAfold, developed at Institute forTheoretical Chemistry at the University of Vienna, using the centroidstructure prediction algorithm (see e.g. A. R. Gruber et al., 2008, Cell106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology27(12): 1151-62). Further algorithms may be found in U.S. applicationSerial No. 61/836,080; Broad Reference BI-2013/004A); incorporatedherein by reference.

In some embodiments, a recombination template is also provided. Arecombination template may be a component of another vector as describedherein, contained in a separate vector, or provided as a separatepolynucleotide. In some embodiments, a recombination template isdesigned to serve as a template in homologous recombination, such aswithin or near a target sequence nicked or cleaved by a nucleicacid-targeting effector protein as a part of a nucleic acid-targetingcomplex. A template polynucleotide may be of any suitable length, suchas about or more than about 10, 15, 20, 25, 50, 75, 100, 150, 200, 500,1000, or more nucleotides in length. In some embodiments, the templatepolynucleotide is complementary to a portion of a polynucleotidecomprising the target sequence. When optimally aligned, a templatepolynucleotide might overlap with one or more nucleotides of a targetsequences (e.g. about or more than about 1, 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 60, 70, 80, 90, 100 or more nucleotides). In someembodiments, when a template sequence and a polynucleotide comprising atarget sequence are optimally aligned, the nearest nucleotide of thetemplate polynucleotide is within about 1, 5, 10, 15, 20, 25, 50, 75,100, 200, 300, 400, 500, 1000, 5000, 10000, or more nucleotides from thetarget sequence.

In some embodiments, the nucleic acid-targeting effector protein is partof a fusion protein comprising one or more heterologous protein domains(e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or moredomains in addition to the nucleic acid-targeting effector protein). Insome embodiments, the CRISPR effector protein/enzyme is part of a fusionprotein comprising one or more heterologous protein domains (e.g. aboutor more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains inaddition to the CRISPR enzyme). A CRISPR effector protein/enzyme fusionprotein may comprise any additional protein sequence, and optionally alinker sequence between any two domains. Examples of protein domainsthat may be fused to an effector protein include, without limitation,epitope tags, reporter gene sequences, and protein domains having one ormore of the following activities: methylase activity, demethylaseactivity, transcription activation activity, transcription repressionactivity, transcription release factor activity, histone modificationactivity, RNA cleavage activity and nucleic acid binding activity.Non-limiting examples of epitope tags include histidine (His) tags, V5tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-Gtags, and thioredoxin (Trx) tags. Examples of reporter genes include,but are not limited to, glutathione-S-transferase (GST), horseradishperoxidase (HRP), chloramphenicol acetyltransferase (CAT)beta-galactosidase, beta-glucuronidase, luciferase, green fluorescentprotein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellowfluorescent protein (YFP), and autofluorescent proteins including bluefluorescent protein (BFP). A nucleic acid-targeting effector protein maybe fused to a gene sequence encoding a protein or a fragment of aprotein that bind DNA molecules or bind other cellular molecules,including but not limited to maltose binding protein (MBP), S-tag, Lex ADNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, andherpes simplex virus (HSV) BP16 protein fusions. Additional domains thatmay form part of a fusion protein comprising a nucleic acid-targetingeffector protein are described in US20110059502, incorporated herein byreference. In some embodiments, a tagged nucleic acid-targeting effectorprotein is used to identify the location of a target sequence.

In some embodiments, a CRISPR enzyme may form a component of aninducible system. The inducible nature of the system would allow forspatiotemporal control of gene editing or gene expression using a formof energy. The form of energy may include but is not limited toelectromagnetic radiation, sound energy, chemical energy and thermalenergy. Examples of inducible system include tetracycline induciblepromoters (Tet-On or Tet-Off), small molecule two-hybrid transcriptionactivations systems (FKBP, ABA, etc), or light inducible systems(Phytochrome, LOV domains, or cryptochrome).In one embodiment, theCRISPR enzyme may be a part of a Light Inducible TranscriptionalEffector (LITE) to direct changes in transcriptional activity in asequence-specific manner. The components of a light may include a CRISPRenzyme, a light-responsive cytochrome heterodimer (e.g. from Arabidopsisthaliana), and a transcriptional activation/repression domain. Furtherexamples of inducible DNA binding proteins and methods for their use areprovided in U.S. 61/736,465 and U.S. 61/721,283 and WO 2014/018423 andU.S. Pat. Nos. 8,889,418, 8,895,308, US20140186919, US20140242700,US20140273234, US20140335620, WO2014093635, which is hereby incorporatedby reference in its entirety.

Delivery

In some aspects, the invention provides methods comprising deliveringone or more polynucleotides, such as or one or more vectors as describedherein, one or more transcripts thereof, and/or one or proteinstranscribed therefrom, to a host cell. In some aspects, the inventionfurther provides cells produced by such methods, and organisms (such asanimals, plants, or fungi) comprising or produced from such cells. Insome embodiments, a nucleic acid-targeting effector protein incombination with (and optionally complexed with) a guide RNA isdelivered to a cell. Conventional viral and non-viral based genetransfer methods can be used to introduce nucleic acids in mammaliancells or target tissues. Such methods can be used to administer nucleicacids encoding components of a nucleic acid-targeting system to cells inculture, or in a host organism. Non-viral vector delivery systemsinclude DNA plasmids, RNA (e.g. a transcript of a vector describedherein), naked nucleic acid, and nucleic acid complexed with a deliveryvehicle, such as a liposome. Viral vector delivery systems include DNAand RNA viruses, which have either episomal or integrated genomes afterdelivery to the cell. For a review of gene therapy procedures, seeAnderson, Science 256:808-813 (1992); Nabel & Felgner, TIBTECH11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon,TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt,Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology andNeuroscience 8:35-36 (1995); Kremer & Perricaudet, British MedicalBulletin 51(1):31-44 (1995); Haddada et al., in Current Topics inMicrobiology and Immunology, Doerfler and Bohm (eds) (1995); and Yu etal., Gene Therapy 1:13-26 (1994).

Methods of non-viral delivery of nucleic acids include lipofection,nucleofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,artificial virions, and agent-enhanced uptake of DNA. Lipofection isdescribed in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355)and lipofection reagents are sold commercially (e.g., Transfectam™ andLipofectin™). Cationic and neutral lipids that are suitable forefficient receptor-recognition lipofection of polynucleotides includethose of Felgner, WO 91/17424; WO 91/16024. Delivery can be to cells(e.g. in vitro or ex vivo administration) or target tissues (e.g. invivo administration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

The use of RNA or DNA viral based systems for the delivery of nucleicacids takes advantage of highly evolved processes for targeting a virusto specific cells in the body and trafficking the viral payload to thenucleus. Viral vectors can be administered directly to patients (invivo) or they can be used to treat cells in vitro, and the modifiedcells may optionally be administered to patients (ex vivo). Conventionalviral based systems could include retroviral, lentivirus, adenoviral,adeno-associated and herpes simplex virus vectors for gene transfer.Integration in the host genome is possible with the retrovirus,lentivirus, and adeno-associated virus gene transfer methods, oftenresulting in long term expression of the inserted transgene.Additionally, high transduction efficiencies have been observed in manydifferent cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system would thereforedepend on the target tissue. Retroviral vectors are comprised ofcis-acting long terminal repeats with packaging capacity for up to 6-10kb of foreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of the vectors, which are then used tointegrate the therapeutic gene into the target cell to provide permanenttransgene expression. Widely used retroviral vectors include those basedupon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV),Simian Immuno deficiency virus (SIV), human immuno deficiency virus(HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700). In applications where transient expression ispreferred, adenoviral based systems may be used. Adenoviral basedvectors are capable of very high transduction efficiency in many celltypes and do not require cell division. With such vectors, high titerand levels of expression have been obtained. This vector can be producedin large quantities in a relatively simple system. Adeno-associatedvirus (“AAV”) vectors may also be used to transduce cells with targetnucleic acids, e.g., in the in vitro production of nucleic acids andpeptides, and for in vivo and ex vivo gene therapy procedures (see,e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368;WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J.Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectorsare described in a number of publications, including U.S. Pat. No.5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985);Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat &Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol.63:03822-3828 (1989).

Options for DNA/RNA or DNA/DNA or RNA/RNA or Protein/RNA

In some embodiments, the components of the CRISPR system may bedelivered in various form, such as combinations of DNA/RNA or RNA/RNA orprotein RNA. For example, the C2c1 or C2c3 may be delivered as aDNA-coding polynucleotide or an RNA-coding polynucleotide or as aprotein. The guide may be delivered may be delivered as a DNA-codingpolynucleotide or an RNA. All possible combinations are envisioned,including mixed forms of delivery.

In some embodiments, all such combinations (DNA/RNA or DNA/DNA orRNA/RNA or protein/RNA).

In some embodiment, when the C2c1 or C2c3 is delivered in protein form,it is possible to pre-assemble same with one or more guide/s.

Nanoclews

Further, the CRISPR system may be delivered using nanoclews, for exampleas described in Sun W et al, Cocoon-like self-degradable DNA nanoclewfor anticancer drug delivery., J Am Chem Soc. 2014 Oct. 22;136(42):14722-5. doi: 10.1021/ja5088024. Epub 2014 Oct. 13; or in Sun Wet al, Self-Assembled DNA Nanoclews for the Efficient Delivery ofCRISPR-Cas9 for Genome Editing., Angew Chem Int Ed Engl. 2015 Oct. 5;54(41):12029-33. doi: 10.1002/anie.201506030. Epub 2015 Aug. 27..

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of immunology, biochemistry,chemistry, molecular biology, microbiology, cell biology, genomics andrecombinant DNA, which are within the skill of the art. See Sambrook,Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2ndedition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel,et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press,Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G.R Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, ALABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)).

Models of Genetic and Euiaenetic Conditions

A method of the invention may be used to create a plant, an animal orcell that may be used to model and/or study genetic or epigeneticconditions of interest, such as a through a model of mutations ofinterest or a disease model. As used herein, “disease” refers to adisease, disorder, or indication in a subject. For example, a method ofthe invention may be used to create an animal or cell that comprises amodification in one or more nucleic acid sequences associated with adisease, or a plant, animal or cell in which the expression of one ormore nucleic acid sequences associated with a disease are altered. Sucha nucleic acid sequence may encode a disease associated protein sequenceor may be a disease associated control sequence. Accordingly, it isunderstood that in embodiments of the invention, a plant, subject,patient, organism or cell can be a non-human subject, patient, organismor cell. Thus, the invention provides a plant, animal or cell, producedby the present methods, or a progeny thereof. The progeny may be a cloneof the produced plant or animal, or may result from sexual reproductionby crossing with other individuals of the same species to introgressfurther desirable traits into their offspring. The cell may be in vivoor ex vivo in the cases of multicellular organisms, particularly animalsor plants. In the instance where the cell is in cultured, a cell linemay be established if appropriate culturing conditions are met andpreferably if the cell is suitably adapted for this purpose (forinstance a stem cell). Bacterial cell lines produced by the inventionare also envisaged. Hence, cell lines are also envisaged.

In some methods, the disease model can be used to study the effects ofmutations on the animal or cell and development and/or progression ofthe disease using measures commonly used in the study of the disease.Alternatively, such a disease model is useful for studying the effect ofa pharmaceutically active compound on the disease.

In some methods, the disease model can be used to assess the efficacy ofa potential gene therapy strategy. That is, a disease-associated gene orpolynucleotide can be modified such that the disease development and/orprogression is inhibited or reduced. In particular, the method comprisesmodifying a disease-associated gene or polynucleotide such that analtered protein is produced and, as a result, the animal or cell has analtered response. Accordingly, in some methods, a genetically modifiedanimal may be compared with an animal predisposed to development of thedisease such that the effect of the gene therapy event may be assessed.

In another embodiment, this invention provides a method of developing abiologically active agent that modulates a cell signaling eventassociated with a disease gene. The method comprises contacting a testcompound with a cell comprising one or more vectors that driveexpression of one or more of a CRISPR enzyme, and a direct repeatsequence linked to a guide sequence; and detecting a change in a readoutthat is indicative of a reduction or an augmentation of a cell signalingevent associated with, e.g., a mutation in a disease gene contained inthe cell.

A cell model or animal model can be constructed in combination with themethod of the invention for screening a cellular function change. Such amodel may be used to study the effects of a genome sequence modified bythe CRISPR complex of the invention on a cellular function of interest.For example, a cellular function model may be used to study the effectof a modified genome sequence on intracellular signaling orextracellular signaling. Alternatively, a cellular function model may beused to study the effects of a modified genome sequence on sensoryperception. In some such models, one or more genome sequences associatedwith a signaling biochemical pathway in the model are modified.

Several disease models have been specifically investigated. Theseinclude de novo autism risk genes CHD8, KATNAL2, and SCN2A; and thesyndromic autism (Angelman Syndrome) gene UBE3A. These genes andresulting autism models are of course preferred, but serve to show thebroad applicability of the invention across genes and correspondingmodels.

An altered expression of one or more genome sequences associated with asignalling biochemical pathway can be determined by assaying for adifference in the mRNA levels of the corresponding genes between thetest model cell and a control cell, when they are contacted with acandidate agent. Alternatively, the differential expression of thesequences associated with a signaling biochemical pathway is determinedby detecting a difference in the level of the encoded polypeptide orgene product.

To assay for an agent-induced alteration in the level of mRNAtranscripts or corresponding polynucleotides, nucleic acid contained ina sample is first extracted according to standard methods in the art.For instance, mRNA can be isolated using various lytic enzymes orchemical solutions according to the procedures set forth in Sambrook etal. (1989), or extracted by nucleic-acid-binding resins following theaccompanying instructions provided by the manufacturers. The mRNAcontained in the extracted nucleic acid sample is then detected byamplification procedures or conventional hybridization assays (e.g.Northern blot analysis) according to methods widely known in the art orbased on the methods exemplified herein.

For purpose of this invention, amplification means any method employinga primer and a polymerase capable of replicating a target sequence withreasonable fidelity. Amplification may be carried out by natural orrecombinant DNA polymerases such as TaqGold™, T7 DNA polymerase, Klenowfragment of E. coli DNA polymerase, and reverse transcriptase. Apreferred amplification method is PCR. In particular, the isolated RNAcan be subjected to a reverse transcription assay that is coupled with aquantitative polymerase chain reaction (RT-PCR) in order to quantify theexpression level of a sequence associated with a signaling biochemicalpathway.

Detection of the gene expression level can be conducted in real time inan amplification assay. In one aspect, the amplified products can bedirectly visualized with fluorescent DNA-binding agents including butnot limited to DNA intercalators and DNA groove binders. Because theamount of the intercalators incorporated into the double-stranded DNAmolecules is typically proportional to the amount of the amplified DNAproducts, one can conveniently determine the amount of the amplifiedproducts by quantifying the fluorescence of the intercalated dye usingconventional optical systems in the art. DNA-binding dye suitable forthis application include SYBR green, SYBR blue, DAPI, propidium iodine,Hoescht, SYBR gold, ethidium bromide, acridines, proflavine, acridineorange, acriflavine, fluorcoumanin, ellipticine, daunomycin,chloroquine, distamycin D, chromomycin, homidium, mithramycin, rutheniumpolypyridyls, anthramycin, and the like.

In another aspect, other fluorescent labels such as sequence specificprobes can be employed in the amplification reaction to facilitate thedetection and quantification of the amplified products. Probe-basedquantitative amplification relies on the sequence-specific detection ofa desired amplified product. It utilizes fluorescent, target-specificprobes (e.g., TaqMan® probes) resulting in increased specificity andsensitivity. Methods for performing probe-based quantitativeamplification are well established in the art and are taught in U.S.Pat. No. 5,210,015.

In yet another aspect, conventional hybridization assays usinghybridization probes that share sequence homology with sequencesassociated with a signaling biochemical pathway can be performed.Typically, probes are allowed to form stable complexes with thesequences associated with a signaling biochemical pathway containedwithin the biological sample derived from the test subject in ahybridization reaction. It will be appreciated by one of skill in theart that where antisense is used as the probe nucleic acid, the targetpolynucleotides provided in the sample are chosen to be complementary tosequences of the antisense nucleic acids. Conversely, where thenucleotide probe is a sense nucleic acid, the target polynucleotide isselected to be complementary to sequences of the sense nucleic acid.

Hybridization can be performed under conditions of various stringency.Suitable hybridization conditions for the practice of the presentinvention are such that the recognition interaction between the probeand sequences associated with a signaling biochemical pathway is bothsufficiently specific and sufficiently stable. Conditions that increasethe stringency of a hybridization reaction are widely known andpublished in the art. See, for example, (Sambrook, et al., (1989);Nonradioactive In Situ Hybridization Application Manual, BoehringerMannheim, second edition). The hybridization assay can be formed usingprobes immobilized on any solid support, including but are not limitedto nitrocellulose, glass, silicon, and a variety of gene arrays. Apreferred hybridization assay is conducted on high-density gene chips asdescribed in U.S. Pat. No. 5,445,934.

For a convenient detection of the probe-target complexes formed duringthe hybridization assay, the nucleotide probes are conjugated to adetectable label. Detectable labels suitable for use in the presentinvention include any composition detectable by photochemical,biochemical, spectroscopic, immunochemical, electrical, optical orchemical means. A wide variety of appropriate detectable labels areknown in the art, which include fluorescent or chemiluminescent labels,radioactive isotope labels, enzymatic or other ligands. In preferredembodiments, one will likely desire to employ a fluorescent label or anenzyme tag, such as digoxigenin, ß-galactosidase, urease, alkalinephosphatase or peroxidase, avidin/biotin complex.

The detection methods used to detect or quantify the hybridizationintensity will typically depend upon the label selected above. Forexample, radiolabels may be detected using photographic film or aphosphorimager. Fluorescent markers may be detected and quantified usinga photodetector to detect emitted light. Enzymatic labels are typicallydetected by providing the enzyme with a substrate and measuring thereaction product produced by the action of the enzyme on the substrate;and finally colorimetric labels are detected by simply visualizing thecolored label.

An agent-induced change in expression of sequences associated with asignaling biochemical pathway can also be determined by examining thecorresponding gene products. Determining the protein level typicallyinvolves a) contacting the protein contained in a biological sample withan agent that specifically bind to a protein associated with a signalingbiochemical pathway; and (b) identifying any agent:protein complex soformed. In one aspect of this embodiment, the agent that specificallybinds a protein associated with a signaling biochemical pathway is anantibody, preferably a monoclonal antibody.

The reaction is performed by contacting the agent with a sample of theproteins associated with a signaling biochemical pathway derived fromthe test samples under conditions that will allow a complex to formbetween the agent and the proteins associated with a signalingbiochemical pathway. The formation of the complex can be detecteddirectly or indirectly according to standard procedures in the art. Inthe direct detection method, the agents are supplied with a detectablelabel and unreacted agents may be removed from the complex; the amountof remaining label thereby indicating the amount of complex formed. Forsuch method, it is preferable to select labels that remain attached tothe agents even during stringent washing conditions. It is preferablethat the label does not interfere with the binding reaction. In thealternative, an indirect detection procedure may use an agent thatcontains a label introduced either chemically or enzymatically. Adesirable label generally does not interfere with binding or thestability of the resulting agent:polypeptide complex. However, the labelis typically designed to be accessible to an antibody for an effectivebinding and hence generating a detectable signal.

A wide variety of labels suitable for detecting protein levels are knownin the art. Non-limiting examples include radioisotopes, enzymes,colloidal metals, fluorescent compounds, bioluminescent compounds, andchemiluminescent compounds.

The amount of agent:polypeptide complexes formed during the bindingreaction can be quantified by standard quantitative assays. Asillustrated above, the formation of agent:polypeptide complex can bemeasured directly by the amount of label remained at the site ofbinding. In an alternative, the protein associated with a signalingbiochemical pathway is tested for its ability to compete with a labeledanalog for binding sites on the specific agent. In this competitiveassay, the amount of label captured is inversely proportional to theamount of protein sequences associated with a signaling biochemicalpathway present in a test sample.

A number of techniques for protein analysis based on the generalprinciples outlined above are available in the art. They include but arenot limited to radioimmunoassays, ELISA (enzyme linked immunoradiometricassays), “sandwich” immunoassays, immunoradiometric assays, in situimmunoassays (using e.g., colloidal gold, enzyme or radioisotopelabels), western blot analysis, immunoprecipitation assays,immunofluorescent assays, and SDS-PAGE.

Antibodies that specifically recognize or bind to proteins associatedwith a signaling biochemical pathway are preferable for conducting theaforementioned protein analyses. Where desired, antibodies thatrecognize a specific type of post-translational modifications (e.g.,signaling biochemical pathway inducible modifications) can be used.Post-translational modifications include but are not limited toglycosylation, lipidation, acetylation, and phosphorylation. Theseantibodies may be purchased from commercial vendors. For example,anti-phosphotyrosine antibodies that specifically recognizetyrosine-phosphorylated proteins are available from a number of vendorsincluding Invitrogen and Perkin Elmer. Antiphosphotyrosine antibodiesare particularly useful in detecting proteins that are differentiallyphosphorylated on their tyrosine residues in response to an ER stress.Such proteins include but are not limited to eukaryotic translationinitiation factor 2 alpha (eIF-2α). Alternatively, these antibodies canbe generated using conventional polyclonal or monoclonal antibodytechnologies by immunizing a host animal or an antibody-producing cellwith a target protein that exhibits the desired post-translationalmodification.

In practicing the subject method, it may be desirable to discern theexpression pattern of an protein associated with a signaling biochemicalpathway in different bodily tissue, in different cell types, and/or indifferent subcellular structures. These studies can be performed withthe use of tissue-specific, cell-specific or subcellular structurespecific antibodies capable of binding to protein markers that arepreferentially expressed in certain tissues, cell types, or subcellularstructures.

An altered expression of a gene associated with a signaling biochemicalpathway can also be determined by examining a change in activity of thegene product relative to a control cell. The assay for an agent-inducedchange in the activity of a protein associated with a signalingbiochemical pathway will dependent on the biological activity and/or thesignal transduction pathway that is under investigation. For example,where the protein is a kinase, a change in its ability to phosphorylatethe downstream substrate(s) can be determined by a variety of assaysknown in the art. Representative assays include but are not limited toimmunoblotting and immunoprecipitation with antibodies such asanti-phosphotyrosine antibodies that recognize phosphorylated proteins.In addition, kinase activity can be detected by high throughputchemiluminescent assays such as AlphaScreen™ (available from PerkinElmer) and eTag™ assay (Chan-Hui, et al. (2003) Clinical Immunology 111:162-174).

Where the protein associated with a signaling biochemical pathway ispart of a signaling cascade leading to a fluctuation of intracellular pHcondition, pH sensitive molecules such as fluorescent pH dyes can beused as the reporter molecules. In another example where the proteinassociated with a signaling biochemical pathway is an ion channel,fluctuations in membrane potential and/or intracellular ionconcentration can be monitored. A number of commercial kits andhigh-throughput devices are particularly suited for a rapid and robustscreening for modulators of ion channels. Representative instrumentsinclude FLIPR™ (Molecular Devices, Inc.) and VIPR (Aurora Biosciences).These instruments are capable of detecting reactions in over 1000 samplewells of a microplate simultaneously, and providing real-timemeasurement and functional data within a second or even a minisecond.

In practicing any of the methods disclosed herein, a suitable vector canbe introduced to a cell or an embryo via one or more methods known inthe art, including without limitation, microinjection, electroporation,sonoporation, biolistics, calcium phosphate-mediated transfection,cationic transfection, liposome transfection, dendrimer transfection,heat shock transfection, nucleofection transfection, magnetofection,lipofection, impalefection, optical transfection, proprietaryagent-enhanced uptake of nucleic acids, and delivery via liposomes,immunoliposomes, virosomes, or artificial virions. In some methods, thevector is introduced into an embryo by microinjection. The vector orvectors may be microinjected into the nucleus or the cytoplasm of theembryo. In some methods, the vector or vectors may be introduced into acell by nucleofection.

The target polynucleotide of a CRISPR complex can be any polynucleotideendogenous or exogenous to the eukaryotic cell. For example, the targetpolynucleotide can be a polynucleotide residing in the nucleus of theeukaryotic cell. The target polynucleotide can be a sequence coding agene product (e.g., a protein) or a non-coding sequence (e.g., aregulatory polynucleotide or a junk DNA).

Examples of target polynucleotides include a sequence associated with asignaling biochemical pathway, e.g., a signaling biochemicalpathway-associated gene or polynucleotide. Examples of targetpolynucleotides include a disease associated gene or polynucleotide. A“disease-associated” gene or polynucleotide refers to any gene orpolynucleotide which is yielding transcription or translation productsat an abnormal level or in an abnormal form in cells derived from adisease-affected tissues compared with tissues or cells of a non diseasecontrol. It may be a gene that becomes expressed at an abnormally highlevel; it may be a gene that becomes expressed at an abnormally lowlevel, where the altered expression correlates with the occurrenceand/or progression of the disease. A disease-associated gene also refersto a gene possessing mutation(s) or genetic variation that is directlyresponsible or is in linkage disequilibrium with a gene(s) that isresponsible for the etiology of a disease. The transcribed or translatedproducts may be known or unknown, and may be at a normal or abnormallevel.

The target polynucleotide of a CRISPR complex can be any polynucleotideendogenous or exogenous to the eukaryotic cell. For example, the targetpolynucleotide can be a polynucleotide residing in the nucleus of theeukaryotic cell. The target polynucleotide can be a sequence coding agene product (e.g., a protein) or a non-coding sequence (e.g., aregulatory polynucleotide or a junk DNA). Without wishing to be bound bytheory, it is believed that the target sequence should be associatedwith a PAM (protospacer adjacent motif); that is, a short sequencerecognized by the CRISPR complex. The precise sequence and lengthrequirements for the PAM differ depending on the CRISPR enzyme used, butPAMs are typically 2-5 base pair sequences adjacent the protospacer(that is, the target sequence) Examples of PAM sequences are given inthe examples section below, and the skilled person will be able toidentify further PAM sequences for use with a given CRISPR enzyme.

The target polynucleotide of a CRISPR complex may include a number ofdisease associated genes and polynucleotides as well as signalingbiochemical pathway-associated genes and polynucleotides as listed inU.S. provisional patent applications 61/736,527 and 61/748,427 bothentitled SYSTEMS METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATIONfiled on Dec. 12, 2012 and Jan. 2, 2013, respectively, and PCTApplication PCT/US2013/074667, entitled DELIVERY, ENGINEERING ANDOPTIMIZATION OF SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCEMANIPULATION AND THERAPEUTIC APPLICATIONS, filed Dec. 12, 2013, thecontents of all of which are herein incorporated by reference in theirentirety.

Examples of target polynucleotides include a sequence associated with asignaling biochemical pathway, e.g., a signaling biochemicalpathway-associated gene or polynucleotide. Examples of targetpolynucleotides include a disease associated gene or polynucleotide. A“disease-associated” gene or polynucleotide refers to any gene orpolynucleotide which is yielding transcription or translation productsat an abnormal level or in an abnormal form in cells derived from adisease-affected tissues compared with tissues or cells of a non diseasecontrol. It may be a gene that becomes expressed at an abnormally highlevel; it may be a gene that becomes expressed at an abnormally lowlevel, where the altered expression correlates with the occurrenceand/or progression of the disease. A disease-associated gene also refersto a gene possessing mutation(s) or genetic variation that is directlyresponsible or is in linkage disequilibrium with a gene(s) that isresponsible for the etiology of a disease. The transcribed or translatedproducts may be known or unknown, and may be at a normal or abnormallevel.

Genome Wide Knock-Out Screening

The CRISPR effector protein complexes described herein can be used toperform efficient and cost effective functional genomic screens. Suchscreens can utilize CRISPR effector protein based genome wide libraries.Such screens and libraries can provide for determining the function ofgenes, cellular pathways genes are involved in, and how any alterationin gene expression can result in a particular biological process. Anadvantage of the present invention is that the CRISPR system avoidsoff-target binding and its resulting side effects. This is achievedusing systems arranged to have a high degree of sequence specificity forthe target DNA. In preferred embodiments of the invention, the CRISPReffector protein complexes are C2C1 or C2c3 effector protein complexes.

In embodiments of the invention, a genome wide library may comprise aplurality of C2C1 or C2c3 guide RNAs, as described herein, comprisingguide sequences that are capable of targeting a plurality of targetsequences in a plurality of genomic loci in a population of eukaryoticcells. The population of cells may be a population of embryonic stem(ES) cells. The target sequence in the genomic locus may be a non-codingsequence. The non-coding sequence may be an intron, regulatory sequence,splice site, 3′ UTR, 5′ UTR, or polyadenylation signal. Gene function ofone or more gene products may be altered by said targeting. Thetargeting may result in a knockout of gene function. The targeting of agene product may comprise more than one guide RNA. A gene product may betargeted by 2, 3, 4, 5, 6, 7, 8, 9, or 10 guide RNAs, preferably 3 to 4per gene. Off-target modifications may be minimized by exploiting thestaggered double strand breaks generated by C2C1 or C2c3 effectorprotein complexes or by utilizing methods analogous to those used inCRISPR-Cas9 systems (See, e.g., DNA targeting specificity of RNA-guidedCas9 nucleases. Hsu, P., Scott, D., Weinstein, J., Ran, FA., Konermann,S., Agarwala, V., Li, Y., Fine, E., Wu, X., Shalem, O., Cradick, T J.,Marraffini, L A., Bao, G., & Zhang, F. Nat Biotechnol.doi:10.1038/nbt.2647 (2013)), incorporated herein by reference. Thetargeting may be of about 100 or more sequences. The targeting may be ofabout 1000 or more sequences. The targeting may be of about 20,000 ormore sequences. The targeting may be of the entire genome. The targetingmay be of a panel of target sequences focused on a relevant or desirablepathway. The pathway may be an immune pathway. The pathway may be a celldivision pathway.

One aspect of the invention comprehends a genome wide library that maycomprise a plurality of C2C1 or C2c3 guide RNAs that may comprise guidesequences that are capable of targeting a plurality of target sequencesin a plurality of genomic loci, wherein said targeting results in aknockout of gene function. This library may potentially comprise guideRNAs that target each and every gene in the genome of an organism.

In some embodiments of the invention the organism or subject is aeukaryote (including mammal including human) or a non-human eukaryote ora non-human animal or a non-human mammal. In some embodiments, theorganism or subject is a non-human animal, and may be an arthropod, forexample, an insect, or may be a nematode. In some methods of theinvention the organism or subject is a plant. In some methods of theinvention the organism or subject is a mammal or a non-human mammal. Anon-human mammal may be for example a rodent (preferably a mouse or arat), an ungulate, or a primate. In some methods of the invention theorganism or subject is algae, including microalgae, or is a fungus.

The knockout of gene function may comprise: introducing into each cellin the population of cells a vector system of one or more vectorscomprising an engineered, non-naturally occurring C2C1 or C2c3 effectorprotein system comprising I. a C2C1 or C2c3 effector protein, and II.one or more guide RNAs, wherein components I and II may be same or ondifferent vectors of the system, integrating components I and II intoeach cell, wherein the guide sequence targets a unique gene in eachcell, wherein the C2C1 or C2c3 effector protein is operably linked to aregulatory element, wherein when transcribed, the guide RNA comprisingthe guide sequence directs sequence-specific binding of the C2C1 or C2c3effector protein system to a target sequence in the genomic loci of theunique gene, inducing cleavage of the genomic loci by the C2C1 or C2c3effector protein, and confirming different knockout mutations in aplurality of unique genes in each cell of the population of cellsthereby generating a gene knockout cell library. The inventioncomprehends that the population of cells is a population of eukaryoticcells, and in a preferred embodiment, the population of cells is apopulation of embryonic stem (ES) cells.

The one or more vectors may be plasmid vectors. The vector may be asingle vector comprising a C2C1 or C2c3 effector protein, a sgRNA, andoptionally, a selection marker into target cells. Not being bound by atheory, the ability to simultaneously deliver a C2C1 or C2c3 effectorprotein and sgRNA through a single vector enables application to anycell type of interest, without the need to first generate cell linesthat express the C2C1 or C2c3 effector protein. The regulatory elementmay be an inducible promoter. The inducible promoter may be adoxycycline inducible promoter. In some methods of the invention theexpression of the guide sequence is under the control of the T7 promoterand is driven by the expression of T7 polymerase. The confirming ofdifferent knockout mutations may be by whole exome sequencing. Theknockout mutation may be achieved in 100 or more unique genes. Theknockout mutation may be achieved in 1000 or more unique genes. Theknockout mutation may be achieved in 20,000 or more unique genes. Theknockout mutation may be achieved in the entire genome. The knockout ofgene function may be achieved in a plurality of unique genes whichfunction in a particular physiological pathway or condition. The pathwayor condition may be an immune pathway or condition. The pathway orcondition may be a cell division pathway or condition.

The invention also provides kits that comprise the genome wide librariesmentioned herein. The kit may comprise a single container comprisingvectors or plasmids comprising the library of the invention. The kit mayalso comprise a panel comprising a selection of unique C2C1 or C2c3effector protein system guide RNAs comprising guide sequences from thelibrary of the invention, wherein the selection is indicative of aparticular physiological condition. The invention comprehends that thetargeting is of about 100 or more sequences, about 1000 or moresequences or about 20,000 or more sequences or the entire genome.Furthermore, a panel of target sequences may be focused on a relevant ordesirable pathway, such as an immune pathway or cell division.

In an additional aspect of the invention, the C2C1 or C2c3 effectorprotein may comprise one or more mutations and may be used as a genericDNA binding protein with or without fusion to a functional domain. Themutations may be artificially introduced mutations or gain- orloss-of-function mutations. The mutations have been characterized asdescribed herein. In one aspect of the invention, the functional domainmay be a transcriptional activation domain, which may be VP64. In otheraspects of the invention, the functional domain may be a transcriptionalrepressor domain, which may be KRAB or SID4X. Other aspects of theinvention relate to the mutated C2C1 or C2c3 effector protein beingfused to domains which include but are not limited to a transcriptionalactivator, repressor, a recombinase, a transposase, a histone remodeler,a demethylase, a DNA methyltransferase, a cryptochrome, a lightinducible/controllable domain or a chemically inducible/controllabledomain. Some methods of the invention can include inducing expression oftargeted genes. In one embodiment, inducing expression by targeting aplurality of target sequences in a plurality of genomic loci in apopulation of eukaryotic cells is by use of a functional domain.

Useful in the practice of the instant invention utilizing C2C1 orC2c3effector protein complexes are methods used in CRISPR-Cas9 systemsand reference is made to:

Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Shalem, O.,Sanjana, N E., Hartenian, E., Shi, X., Scott, D A., Mikkelson, T.,Heckl, D., Ebert, B L., Root, D E., Doench, J G., Zhang, F. ScienceDecember 12. (2013). [Epub ahead of print]; Published in final editedform as: Science. 2014 Jan. 3; 343(6166): 84-87.

Shalem et al. involves a new way to interrogate gene function on agenome-wide scale. Their studies showed that delivery of a genome-scaleCRISPR-Cas9 knockout (GeCKO) library targeted 18,080 genes with 64,751unique guide sequences enabled both negative and positive selectionscreening in human cells. First, the authors showed use of the GeCKOlibrary to identify genes essential for cell viability in cancer andpluripotent stem cells. Next, in a melanoma model, the authors screenedfor genes whose loss is involved in resistance to vemurafenib, atherapeutic that inhibits mutant protein kinase BRAF. Their studiesshowed that the highest-ranking candidates included previously validatedgenes NF1 and MED12 as well as novel hitsNF2, CUL3, TADA2B, and TADA1.The authors observed a high level of consistency between independentguide RNAs targeting the same gene and a high rate of hit confirmation,and thus demonstrated the promise of genome-scale screening with Cas9.

Reference is also made to US patent publication number US20140357530;and PCT Patent Publication WO2014093701, hereby incorporated herein byreference. Reference is also made to NIH Press Release of Oct. 22, 2015entitled, “Researchers identify potential alternative to CRISPR-Casgenome editing tools: New Cas enzymes shed light on evolution ofCRISPR-Cas systems, which is incorporated by reference.

Functional Alteration and Screening

In another aspect, the present invention provides for a method offunctional evaluation and screening of genes. The use of the CRISPRsystem of the present invention to precisely deliver functional domains,to activate or repress genes or to alter epigenetic state by preciselyaltering the methylation site on a specific locus of interest, can bewith one or more guide RNAs applied to a single cell or population ofcells or with a library applied to genome in a pool of cells ex vivo orin vivo comprising the administration or expression of a librarycomprising a plurality of guide RNAs (sgRNAs) and wherein the screeningfurther comprises use of a C2C1 or C2c3 effector protein, wherein theCRISPR complex comprising the C2C1 or C2c3 effector protein is modifiedto comprise a heterologous functional domain. In an aspect the inventionprovides a method for screening a genome comprising the administrationto a host or expression in a host in vivo of a library. In an aspect theinvention provides a method as herein discussed further comprising anactivator administered to the host or expressed in the host. In anaspect the invention provides a method as herein discussed wherein theactivator is attached to a C2C1 or C2c3 effector protein. In an aspectthe invention provides a method as herein discussed wherein theactivator is attached to the N terminus or the C terminus of the C2C1 orC2c3 effector protein. In an aspect the invention provides a method asherein discussed wherein the activator is attached to a sgRNA loop. Inan aspect the invention provides a method as herein discussed furthercomprising a repressor administered to the host or expressed in thehost. In an aspect the invention provides a method as herein discussed,wherein the screening comprises affecting and detecting gene activation,gene inhibition, or cleavage in the locus.

In an aspect, the invention provides efficient on-target activity andminimizes off target activity. In an aspect, the invention providesefficient on-target cleavage by C2C1 or C2c3 effector protein andminimizes off-target cleavage by the C2C1 or C2c3 effector protein. Inan aspect, the invention provides guide specific binding of C2C1 or C2c3effector protein at a gene locus without DNA cleavage. Accordingly, inan aspect, the invention provides target-specific gene regulation. In anaspect, the invention provides guide specific binding of C2C1 or C2c3effector protein at a gene locus without DNA cleavage. Accordingly, inan aspect, the invention provides for cleavage at one gene locus andgene regulation at a different gene locus using a single C2C1 or C2c3effector protein. In an aspect, the invention provides orthogonalactivation and/or inhibition and/or cleavage of multiple targets usingone or more C2C1 or C2c3 effector protein and/or enzyme.

In an aspect the invention provides a method as herein discussed,wherein the host is a eukaryotic cell. In an aspect the inventionprovides a method as herein discussed, wherein the host is a mammaliancell. In an aspect the invention provides a method as herein discussed,wherein the host is a non-human eukaryote. In an aspect the inventionprovides a method as herein discussed, wherein the non-human eukaryoteis a non-human mammal. In an aspect the invention provides a method asherein discussed, wherein the non-human mammal is a mouse. An aspect theinvention provides a method as herein discussed comprising the deliveryof the C2C1 or C2c3 effector protein complexes or component(s) thereofor nucleic acid molecule(s) coding therefor, wherein said nucleic acidmolecule(s) are operatively linked to regulatory sequence(s) andexpressed in vivo. In an aspect the invention provides a method asherein discussed wherein the expressing in vivo is via a lentivirus, anadenovirus, or an AAV. In an aspect the invention provides a method asherein discussed wherein the delivery is via a particle, a nanoparticle,a lipid or a cell penetrating peptide (CPP).

In an aspect the invention provides a pair of CRISPR complexescomprising C2C1 or C2c3 effector protein, each comprising a guide RNA(sgRNA) comprising a guide sequence capable of hybridizing to a targetsequence in a genomic locus of interest in a cell, wherein at least oneloop of each sgRNA is modified by the insertion of distinct RNAsequence(s) that bind to one or more adaptor proteins, and wherein theadaptor protein is associated with one or more functional domains,wherein each sgRNA of each C2C1 or C2c3 effector protein complexcomprises a functional domain having a DNA cleavage activity. In anaspect the invention provides paired C2C1 or C2c3 effector proteincomplexes as herein-discussed, wherein the DNA cleavage activity is dueto a Fok1 nuclease.

In an aspect the invention provides a method for cutting a targetsequence in a genomic locus of interest comprising delivery to a cell ofthe C2C1 or C2c3 effector protein complexes or component(s) thereof ornucleic acid molecule(s) coding therefor, wherein said nucleic acidmolecule(s) are operatively linked to regulatory sequence(s) andexpressed in vivo. In an aspect the invention provides a method asherein-discussed wherein the delivery is via a lentivirus, anadenovirus, or an AAV. In an aspect the invention provides a method asherein-discussed or paired C2C1 or C2c3 effector protein complexes asherein-discussed wherein the target sequence for a first complex of thepair is on a first strand of double stranded DNA and the target sequencefor a second complex of the pair is on a second strand of doublestranded DNA. In an aspect the invention provides a method asherein-discussed or paired C2C1 or C2c3 effector protein complexes asherein-discussed wherein the target sequences of the first and secondcomplexes are in proximity to each other such that the DNA is cut in amanner that facilitates homology directed repair. In an aspect a hereinmethod can further include introducing into the cell template DNA. In anaspect a herein method or herein paired C2C1 or C2c3 effector proteincomplexes can involve wherein each C2C1 or C2c3 effector protein complexhas a C2C1 or C2c3 effector enzyme that is mutated such that it has nomore than about 5% of the nuclease activity of the C2C1 or C2c3 effectorenzyme that is not mutated.

In an aspect the invention provides a library, method or complex asherein-discussed wherein the sgRNA is modified to have at least onenon-coding functional loop, e.g., wherein the at least one non-codingfunctional loop is repressive; for instance, wherein the at least onenon-coding functional loop comprises Alu.

In one aspect, the invention provides a method for altering or modifyingexpression of a gene product. The said method may comprise introducinginto a cell containing and expressing a DNA molecule encoding the geneproduct an engineered, non-naturally occurring CRISPR system comprisinga C2C1 or C2c3 effector protein and guide RNA that targets the DNAmolecule, whereby the guide RNA targets the DNA molecule encoding thegene product and the C2C1 or C2c3 effector protein cleaves the DNAmolecule encoding the gene product, whereby expression of the geneproduct is altered; and, wherein the C2C1 or C2c3 effector protein andthe guide RNA do not naturally occur together. The invention comprehendsthe guide RNA comprising a guide sequence linked to a direct repeatsequence. The invention further comprehends the C2C1 or C2c3 effectorprotein being codon optimized for expression in a Eukaryotic cell. In apreferred embodiment the Eukaryotic cell is a mammalian cell and in amore preferred embodiment the mammalian cell is a human cell. In afurther embodiment of the invention, the expression of the gene productis decreased.

In some embodiments, one or more functional domains are associated withthe C2C1 or C2c3 effector protein. In some embodiments, one or morefunctional domains are associated with an adaptor protein, for exampleas used with the modified guides of Konnerman et al. (Nature 517,583-588, 29 Jan. 2015). In some embodiments, one or more functionaldomains are associated with an dead sgRNA (dRNA). In some embodiments, adRNA complex with active C2C1 or C2c3 effector protein directs generegulation by a functional domain at on gene locus while an sgRNAdirects DNA cleavage by the active C2C1 or C2c3 effector protein atanother locus, for example as described analogously in CRISPR-Cas9systems by Dahlman et al., ‘Orthogonal gene control with a catalyticallyactive Cas9 nuclease’ (in press). In some embodiments, dRNAs areselected to maximize selectivity of regulation for a gene locus ofinterest compared to off-target regulation. In some embodiments, dRNAsare selected to maximize target gene regulation and minimize targetcleavage.

For the purposes of the following discussion, reference to a functionaldomain could be a functional domain associated with the C2C1 or C2c3effector protein or a functional domain associated with the adaptorprotein.

In some embodiments, the one or more functional domains is an NLS(Nuclear Localization Sequence) or an NES (Nuclear Export Signal). Insome embodiments, the one or more functional domains is atranscriptional activation domain comprises VP64, p65, MyoD1, HSF1, RTA,SET7/9 and a histone acetyltransferase. Other references herein toactivation (or activator) domains in respect of those associated withthe CRISPR enzyme include any known transcriptional activation domainand specifically VP64, p65, MyoD1, HSF1, RTA, SET7/9 or a histoneacetyltransferase.

In some embodiments, the one or more functional domains is atranscriptional repressor domain. In some embodiments, thetranscriptional repressor domain is a KRAB domain. In some embodiments,the transcriptional repressor domain is a NuE domain, NcoR domain, SIDdomain or a SID4X domain.

In some embodiments, the one or more functional domains have one or moreactivities comprising methylase activity, demethylase activity,transcription activation activity, transcription repression activity,transcription release factor activity, histone modification activity,RNA cleavage activity, DNA cleavage activity, DNA integration activityor nucleic acid binding activity.

Histone modifying domains are also preferred in some embodiments.Exemplary histone modifying domains are discussed below. Transposasedomains, HR (Homologous Recombination) machinery domains, recombinasedomains, and/or integrase domains are also preferred as the presentfunctional domains. In some embodiments, DNA integration activityincludes HR machinery domains, integrase domains, recombinase domainsand/or transposase domains. Histone acetyltransferases are preferred insome embodiments.

In some embodiments, the DNA cleavage activity is due to a nuclease. Insome embodiments, the nuclease comprises a Fok1 nuclease. See, “DimericCRISPR RNA-guided FokI nucleases for highly specific genome editing”,Shengdar Q. Tsai, Nicolas Wyvekens, Cyd Khayter, Jennifer A. Foden,Vishal Thapar, Deepak Reyon, Mathew J. Goodwin, Martin J. Aryee, J.Keith Joung Nature Biotechnology 32(6): 569-77 (2014), relates todimeric RNA-guided FokI Nucleases that recognize extended sequences andcan edit endogenous genes with high efficiencies in human cells.

In some embodiments, the one or more functional domains is attached tothe C2C1 or C2c3 effector protein so that upon binding to the sgRNA andtarget the functional domain is in a spatial orientation allowing forthe functional domain to function in its attributed function.

In some embodiments, the one or more functional domains is attached tothe adaptor protein so that upon binding of the C2C1 or C2c3 effectorprotein to the sgRNA and target, the functional domain is in a spatialorientation allowing for the functional domain to function in itsattributed function.

In an aspect the invention provides a composition as herein discussedwherein the one or more functional domains is attached to the C2C1 orC2c3 effector protein or adaptor protein via a linker, optionally aGlySer linker, as discussed herein.

Endogenous transcriptional repression is often mediated by chromatinmodifying enzymes such as histone methyltransferases (HMTs) anddeacetylases (HDACs). Repressive histone effector domains are known andan exemplary list is provided below. In the exemplary table, preferencewas given to proteins and functional truncations of small size tofacilitate efficient viral packaging (for instance via AAV). In general,however, the domains may include HDACs, histone methyltransferases(HMTs), and histone acetyltransferase (HAT) inhibitors, as well as HDACand HMT recruiting proteins. The functional domain may be or include, insome embodiments, HDAC Effector Domains, HDAC Recruiter EffectorDomains, Histone Methyltransferase (HMT) Effector Domains, HistoneMethyltransferase (HMT) Recruiter Effector Domains, or HistoneAcetyltransferase Inhibitor Effector Domains.

TABLE 3 HDAC Effector Domains Substrate Full Selected Final Subtype/ (ifModification size truncation size Catalytic Complex Name known) (ifknown) Organism (aa) (aa) (aa) domain HDAC I HDAC8 — — X. laevis 3251-325 325 1-272: HDAC HDAC I RPD3 — — S. cerevisiae 433 19-340  32219-331: (Vannier) HDAC HDAC MesoLo4 — — M. loti 300 1-300 300 — IV(Gregoretti) HDAC HDAC11 — — H. sapiens 347 1-347 347 14-326: IV (Gao)HDAC HD2 HDT1 — — A. thaliana 245 1-211 211 — (Wu) SIRT I SIRT3 H3K9Ac —H. sapiens 399 143-399 257 126-382: H4K16Ac (Scher) SIRT H3K56Ac SIRT IHST2 — — C. albicans 331 1-331 331 — (Hnisz) SIRT I CobB — — E. coli 2421-242 242 — (K12) (Landry) SIRT I HST2 — — S. cerevisiae 357 8-298 291 —(Wilson) SIRT SIRT5 H4K8Ac — H. sapiens 310 37-310  274 41-309: IIIH4K16Ac (Gertz) SIRT SIRT Sir2A — — P. falciparum 273 1-273 273 19-273III (Zhu) SIRT SIRT SIRT6 H3K9Ac — H. sapiens 355 1-289 289 35-274: IVH3K56Ac (Tennen) SIRT

Accordingly, the repressor domains of the present invention may beselected from histone methyltransferases (HMTs), histone deacetylases(HDACs), histone acetyltransferase (HAT) inhibitors, as well as HDAC andHMT recruiting proteins.

The HDAC domain may be any of those in the table above, namely: HDAC8,RPD3, MesoLo4, HDAC11, HDT1, SIRT3, HST2, CobB, HST2, SIRT5, Sir2A, orSIRT6.

In some embodiment, the functional domain may be a HDAC RecruiterEffector Domain. Preferred examples include those in the Table below,namely MeCP2, MBD2b, Sin3a, NcoR, SALL1, RCOR1. NcoR is exemplified inthe present Examples and, although preferred, it is envisaged thatothers in the class will also be useful.

TABLE 4 Table of HDAC Recruiter Effector Domains Substrate Full SelectedFinal Subtype/ (if Modification size truncation size Catalytic ComplexName known) (if known) Organism (aa) (aa) (aa) domain Sin3a MeCP2 — — R.norvegicus 492 207-492 286 — (Nan) Sin3a MBD2b — — H. sapiens 262 45-262 218 — (Boeke) Sin3a Sin3a — — H. sapiens 1273 524-851 328627-829: (Laherty) HDAC1 interaction NcoR NcoR — — H. sapiens 2440420-488 69 — (Zhang) NuRD SALL1 — — M. musculus 1322  1-93 93 —(Lauberth) CoREST RCOR1 — — H. sapiens 482  81-300 220 — (Gu, Ouyang)

In some embodiment, the functional domain may be a Methyltransferase(HMT) Effector Domain. Preferred examples include those in the Tablebelow, namely NUE, vSET, EHMT2/G9A, SUV39H1, dim-5, KYP, SUVR4, SET4,SET, SETD8, and TgSET8. NUE is exemplified in the present Examples and,although preferred, it is envisaged that others in the class will alsobe useful.

TABLE 5 Table of Histone Methyltransferase (HMT) Effector DomainsSubstrate Modification Full Selected Subtype/ (if (if size truncationFinal size Catalytic Complex Name known) known) Organism (aa) (aa) (aa)domain SET NUE H2B, — C. trachomatis 219  1-219 219 — H3, H4 (Pennini)SET vSET — H3K27me3 P. bursaria 119  1-119 119 4-112: chlorella(Mujtaba) SET2 virus SUV39 EHMT2/ H1.4K2, H3K9me1/ M. musculus 1263 969-1263 295 1025-1233: family G9A H3K9, 2, (Tachibana) preSET, H3K27H1K25me1 SET, postSET SUV39 SUV39H1 — H3K9me2/3 H. sapiens 412  79-412334 172-412: (Snowden) preSET, SET, postSET Suvar3-9 dim-5 — H3K9me3 N.crassa 331  1-331 331 77-331: (Rathert) preSET, SET, postSET Suvar3-9KYP — H3K9me1/2 A. thaliana 624 335-601 267 — (SUVH (Jackson) subfamily)Suvar3-9 SUVR4 H3K9me1 H3K9me2/3 A. thaliana 492 180-492 313 192-462:(SUVR (Thorstensen) preSET, subfamily) SET, postSET Suvar4- SET4 —H4K20me3 C. elegans 288  1-288 288 — 20 (Vielle) SET8 SET1 — H4K20me1 C.elegans 242  1-242 242 — (Vielle) SET8 SETD8 — H4K20me1 H. sapiens 393185-393 209 256-382: (Couture) SET SET8 TgSET8 — H4K20me1/ T. gondii1893 1590-1893 304 1749-1884: 2/3 (Sautel) SET

In some embodiment, the functional domain may be a HistoneMethyltransferase (HMT) Recruiter Effector Domain. Preferred examplesinclude those in the Table below, namely Hp1a, PHF19, and NIPP1.

TABLE 6 Table of Histone Methyltransferase (HMT) Recruiter EffectorDomains Substrate Full Selected Subtype/ (if Modification sizetruncation Final size Catalytic Complex Name known) (if known) Organism(aa) (aa) (aa) domain — Hp1a — H3K9me3 M. musculus 191 73-191 119121-179: (Hathaway) chromoshadow — PHF19 — H3K27me3 H. sapiens 580(1-250) + 335 163-250: GGSG (Ballare) PHD2 linker + (500-580) — NIPP1 —H3K27me3 H. sapiens 351  1-329 329 310-329: (Jin) EED

In some embodiment, the functional domain may be HistoneAcetyltransferase Inhibitor Effector Domain. Preferred examples includeSET/TAF-1β listed in the Table below.

TABLE 7 Table of Histone Acetyltransferase Inhibitor Effector DomainsSubstrate Full Selected Final Subtype/ (if Modification size truncationsize Catalytic Complex Name known) (if known) Organism (aa) (aa) (aa)domain — SET/TAF- — — M. musculus 289 1-289 289 — 1β (Cervoni)

It is also preferred to target endogenous (regulatory) control elements(such as enhancers and silencers) in addition to a promoter orpromoter-proximal elements. Thus, the invention can also be used totarget endogenous control elements (including enhancers and silencers)in addition to targeting of the promoter. These control elements can belocated upstream and downstream of the transcriptional start site (TSS),starting from 200 bp from the TSS to 100 kb away. Targeting of knowncontrol elements can be used to activate or repress the gene ofinterest. In some cases, a single control element can influence thetranscription of multiple target genes. Targeting of a single controlelement could therefore be used to control the transcription of multiplegenes simultaneously.

Targeting of putative control elements on the other hand (e.g. by tilingthe region of the putative control element as well as 200 bp up to 100kB around the element) can be used as a means to verify such elements(by measuring the transcription of the gene of interest) or to detectnovel control elements (e.g. by tiling 100 kb upstream and downstream ofthe TSS of the gene of interest). In addition, targeting of putativecontrol elements can be useful in the context of understanding geneticcauses of disease. Many mutations and common SNP variants associatedwith disease phenotypes are located outside coding regions. Targeting ofsuch regions with either the activation or repression systems describedherein can be followed by readout of transcription of either a) a set ofputative targets (e.g. a set of genes located in closest proximity tothe control element) or b) whole-transcriptome readout by e.g. RNAseq ormicroarray. This would allow for the identification of likely candidategenes involved in the disease phenotype. Such candidate genes could beuseful as novel drug targets.

Histone acetyltransferase (HAT) inhibitors are mentioned herein.However, an alternative in some embodiments is for the one or morefunctional domains to comprise an acetyltransferase, preferably ahistone acetyltransferase. These are useful in the field of epigenomics,for example in methods of interrogating the epigenome. Methods ofinterrogating the epigenome may include, for example, targetingepigenomic sequences. Targeting epigenomic sequences may include theguide being directed to an epigenomic target sequence. Epigenomic targetsequence may include, in some embodiments, include a promoter, silenceror an enhancer sequence.

Use of a functional domain linked to a C2C1 or C2c3 effector protein asdescribed herein, preferably a dead-C2C1 or C2c3 effector protein, morepreferably a dead-Aac C2C1 or C2c3 effector protein, to targetepigenomic sequences can be used to activate or repress promoters,silencer or enhancers.

Examples of acetyltransferases are known but may include, in someembodiments, histone acetyltransferases. In some embodiments, thehistone acetyltransferase may comprise the catalytic core of the humanacetyltransferase p300 (Gerbasch & Reddy, Nature Biotech 6 Apr. 2015).

In some preferred embodiments, the functional domain is linked to adead-C2C1 or C2c3 effector protein to target and activate epigenomicsequences such as promoters or enhancers. One or more guides directed tosuch promoters or enhancers may also be provided to direct the bindingof the CRISPR enzyme to such promoters or enhancers.

The term “associated with” is used here in relation to the associationof the functional domain to the C2C1 or C2c3 effector protein or theadaptor protein. It is used in respect of how one molecule ‘associates’with respect to another, for example between an adaptor protein and afunctional domain, or between the C2C1 or C2c3 effector protein and afunctional domain. In the case of such protein-protein interactions,this association may be viewed in terms of recognition in the way anantibody recognizes an epitope. Alternatively, one protein may beassociated with another protein via a fusion of the two, for instanceone subunit being fused to another subunit. Fusion typically occurs byaddition of the amino acid sequence of one to that of the other, forinstance via splicing together of the nucleotide sequences that encodeeach protein or subunit. Alternatively, this may essentially be viewedas binding between two molecules or direct linkage, such as a fusionprotein. In any event, the fusion protein may include a linker betweenthe two subunits of interest (i.e. between the enzyme and the functionaldomain or between the adaptor protein and the functional domain). Thus,in some embodiments, the C2C1 or C2c3 effector protein or adaptorprotein is associated with a functional domain by binding thereto. Inother embodiments, the C2C1 or C2c3 effector protein or adaptor proteinis associated with a functional domain because the two are fusedtogether, optionally via an intermediate linker.

Attachment of a functional domain or fusion protein can be via a linker,e.g., a flexible glycine-serine (GlyGlyGlySer) (SEQ ID NO: 17) or(GGGS)₃ (SEQ ID NO: 34) or a rigid alpha-helical linker such as(Ala(GluAlaAlaAlaLys)Ala) (SEQ ID NO: 35). Linkers such as (GGGGS)3 (SEQID NO: 18) are preferably used herein to separate protein or peptidedomains. (GGGGS)₃ (SEQ ID NO: 18) is preferable because it is arelatively long linker (15 amino acids). The glycine residues are themost flexible and the serine residues enhance the chance that the linkeris on the outside of the protein. (GGGGS)₆ (SEQ ID NO: 19) (GGGGS)₉ (SEQID NO: 20) or (GGGGS)₁₂ (SEQ ID NO: 21) may preferably be used asalternatives. Other preferred alternatives are (GGGGS)i (SEQ ID NO: 22),(GGGGS)₂ (SEQ ID NO: 23), (GGGGS)₄ (SEQ ID NO: 24), (GGGGS)₅ (SEQ ID NO:25), (GGGGS)₇ (SEQ ID NO: 26), (GGGGS)₈ (SEQ ID NO: 27), (GGGGS)₁₀ (SEQID NO: 28), or (GGGGS)ii (SEQ ID NO: 29). Alternative linkers areavailable, but highly flexible linkers are thought to work best to allowfor maximum opportunity for the 2 parts of the C2c1 or C2c3 to cometogether and thus reconstitute C2c1 or C2c3 activity. One alternative isthat the NLS of nucleoplasmin can be used as a linker. For example, alinker can also be used between the C2c1 and any functional domain orbetween the C2c3 and any functional domain. Again, a (GGGGS)3 (SEQ IDNO: 18) linker may be used here (or the 6 (SEQ ID NO: 19), 9 (SEQ ID NO:20), or 12 (SEQ ID NO: 21) repeat versions therefore) or the NLS ofnucleoplasmin can be used as a linker between C2c1 and the functionaldomain or between C2c3 and the functional domain.

Saturating Mutagenesis

The C2C1 or C2c3 effector protein system(s) described herein can be usedto perform saturating or deep scanning mutagenesis of genomic loci inconjunction with a cellular phenotype—for instance, for determiningcritical minimal features and discrete vulnerabilities of functionalelements required for gene expression, drug resistance, and reversal ofdisease. By saturating or deep scanning mutagenesis is meant that everyor essentially every DNA base is cut within the genomic loci. A libraryof C2C1 or C2c3 effector protein guide RNAs may be introduced into apopulation of cells. The library may be introduced, such that each cellreceives a single guide RNA (sgRNA). In the case where the library isintroduced by transduction of a viral vector, as described herein, a lowmultiplicity of infection (MOI) is used. The library may include sgRNAstargeting every sequence upstream of a (protospacer adjacent motif)(PAM) sequence in a genomic locus. The library may include at least 100non-overlapping genomic sequences upstream of a PAM sequence for every1000 base pairs within the genomic locus. The library may include sgRNAstargeting sequences upstream of at least one different PAM sequence. TheC2C1 or C2c3 effector protein systems may include more than one C2C1 orC2c3 protein. Any C2C1 or C2c3 effector protein as described herein,including orthologues or engineered C2C1 or C2c3 effector proteins thatrecognize different PAM sequences may be used. The frequency of offtarget sites for a sgRNA may be less than 500. Off target scores may begenerated to select sgRNAs with the lowest off target sites. Anyphenotype determined to be associated with cutting at a sgRNA targetsite may be confirmed by using sgRNAs targeting the same site in asingle experiment. Validation of a target site may also be performed byusing a modified C2C1 or C2c3 effector protein, as described herein, andtwo sgRNAs targeting the genomic site of interest. Not being bound by atheory, a target site is a true hit if the change in phenotype isobserved in validation experiments.

The genomic loci may include at least one continuous genomic region. Theat least one continuous genomic region may comprise up to the entiregenome. The at least one continuous genomic region may comprise afunctional element of the genome. The functional element may be within anon-coding region, coding gene, intronic region, promoter, or enhancer.The at least one continuous genomic region may comprise at least 1 kb,preferably at least 50 kb of genomic DNA. The at least one continuousgenomic region may comprise a transcription factor binding site. The atleast one continuous genomic region may comprise a region of DNase Ihypersensitivity. The at least one continuous genomic region maycomprise a transcription enhancer or repressor element. The at least onecontinuous genomic region may comprise a site enriched for an epigeneticsignature. The at least one continuous genomic DNA region may comprisean epigenetic insulator. The at least one continuous genomic region maycomprise two or more continuous genomic regions that physicallyinteract. Genomic regions that interact may be determined by ‘4Ctechnology’. 4C technology allows the screening of the entire genome inan unbiased manner for DNA segments that physically interact with a DNAfragment of choice, as is described in Zhao et al. ((2006) Nat Genet 38,1341-7) and in U.S. Pat. No. 8,642,295, both incorporated herein byreference in its entirety. The epigenetic signature may be histoneacetylation, histone methylation, histone ubiquitination, histonephosphorylation, DNA methylation, or a lack thereof.

The C2C1 or C2c3 effector protein system(s) for saturating or deepscanning mutagenesis can be used in a population of cells. The C2C1 orC2c3 effector protein system(s) can be used in eukaryotic cells,including but not limited to mammalian and plant cells. The populationof cells may be prokaryotic cells. The population of eukaryotic cellsmay be a population of embryonic stem (ES) cells, neuronal cells,epithelial cells, immune cells, endocrine cells, muscle cells,erythrocytes, lymphocytes, plant cells, or yeast cells.

In one aspect, the present invention provides for a method of screeningfor functional elements associated with a change in a phenotype. Thelibrary may be introduced into a population of cells that are adapted tocontain a C2C1 or C2c3 effector protein. The cells may be sorted into atleast two groups based on the phenotype. The phenotype may be expressionof a gene, cell growth, or cell viability. The relative representationof the guide RNAs present in each group are determined, whereby genomicsites associated with the change in phenotype are determined by therepresentation of guide RNAs present in each group. The change inphenotype may be a change in expression of a gene of interest. The geneof interest may be upregulated, downregulated, or knocked out. The cellsmay be sorted into a high expression group and a low expression group.The population of cells may include a reporter construct that is used todetermine the phenotype. The reporter construct may include a detectablemarker. Cells may be sorted by use of the detectable marker.

In another aspect, the present invention provides for a method ofscreening for genomic sites associated with resistance to a chemicalcompound. The chemical compound may be a drug or pesticide. The librarymay be introduced into a population of cells that are adapted to containa C2C1 or C2c3 effector protein, wherein each cell of the populationcontains no more than one guide RNA; the population of cells are treatedwith the chemical compound; and the representation of guide RNAs aredetermined after treatment with the chemical compound at a later timepoint as compared to an early time point, whereby genomic sitesassociated with resistance to the chemical compound are determined byenrichment of guide RNAs. Representation of sgRNAs may be determined bydeep sequencing methods.

Useful in the practice of the instant invention utilizing C2C1 orC2c3effector protein complexes are methods used in CRISPR-Cas9 systemsand reference is made to the article entitled BCL11A enhancer dissectionby Cas9-mediated in situ saturating mutagenesis. Canver, M. C., Smith,E. C., Sher, F., Pinello, L., Sanjana, N. E., Shalem, O., Chen, D. D.,Schupp, P. G., Vinjamur, D. S., Garcia, S. P., Luc, S., Kurita, R.,Nakamura, Y., Fujiwara, Y., Maeda, T., Yuan, G., Zhang, F., Orkin, S.H., & Bauer, D. E. DOI:10.1038/nature15521, published online Sep. 16,2015, the article is herein incorporated by reference and discussedbriefly below:

Canver et al. involves novel pooled CRISPR-Cas9 guide RNA libraries toperform in situ saturating mutagenesis of the human and mouse BCL11Aerythroid enhancers previously identified as an enhancer associated withfetal hemoglobin (HbF) level and whose mouse ortholog is necessary forerythroid BCL11A expression. This approach revealed critical minimalfeatures and discrete vulnerabilities of these enhancers. Throughediting of primary human progenitors and mouse transgenesis, the authorsvalidated the BCL11A erythroid enhancer as a target for HbF reinduction.The authors generated a detailed enhancer map that informs therapeuticgenome editing.

Method of Using C201 or C2C3 Systems to Modify a Cell or Organism

The invention in some embodiments comprehends a method of modifying acell or organism. The cell may be a prokaryotic cell or a eukaryoticcell. The cell may be a mammalian cell. The mammalian cell many be anon-human primate, bovine, porcine, rodent or mouse cell. The cell maybe a non-mammalian eukaryotic cell such as poultry, fish or shrimp. Thecell may also be a plant cell. The plant cell may be of a crop plantsuch as cassava, corn, sorghum, wheat, or rice. The plant cell may alsobe of an algae, tree or vegetable. The modification introduced to thecell by the present invention may be such that the cell and progeny ofthe cell are altered for improved production of biologic products suchas an antibody, starch, alcohol or other desired cellular output. Themodification introduced to the cell by the present invention may be suchthat the cell and progeny of the cell include an alteration that changesthe biologic product produced.

The system may comprise one or more different vectors. In an aspect ofthe invention, the effector protein is codon optimized for expressionthe desired cell type, preferentially a eukaryotic cell, preferably amammalian cell or a human cell.

Packaging cells are typically used to form virus particles that arecapable of infecting a host cell. Such cells include 293 cells, whichpackage adenovirus, and ψ2 cells or PA317 cells, which packageretrovirus. Viral vectors used in gene therapy are usually generated byproducing a cell line that packages a nucleic acid vector into a viralparticle. The vectors typically contain the minimal viral sequencesrequired for packaging and subsequent integration into a host, otherviral sequences being replaced by an expression cassette for thepolynucleotide(s) to be expressed. The missing viral functions aretypically supplied in trans by the packaging cell line. For example, AAVvectors used in gene therapy typically only possess ITR sequences fromthe AAV genome which are required for packaging and integration into thehost genome. Viral DNA is packaged in a cell line, which contains ahelper plasmid encoding the other AAV genes, namely rep and cap, butlacking ITR sequences. The cell line may also be infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV. Additionalmethods for the delivery of nucleic acids to cells are known to thoseskilled in the art. See, for example, US20030087817, incorporated hereinby reference.

Delivery

The invention involves at least one component of the CRISPR complex,e.g., RNA, delivered via at least one nanoparticle complex. In someaspects, the invention provides methods comprising delivering one ormore polynucleotides, such as or one or more vectors as describedherein, one or more transcripts thereof, and/or one or proteinstranscribed therefrom, to a host cell. In some aspects, the inventionfurther provides cells produced by such methods, and animals comprisingor produced from such cells. In some embodiments, a CRISPR enzyme incombination with (and optionally complexed with) a guide sequence isdelivered to a cell. Conventional viral and non-viral based genetransfer methods can be used to introduce nucleic acids in mammaliancells or target tissues. Such methods can be used to administer nucleicacids encoding components of a CRISPR system to cells in culture, or ina host organism. Non-viral vector delivery systems include DNA plasmids,RNA (e.g. a transcript of a vector described herein), naked nucleicacid, and nucleic acid complexed with a delivery vehicle, such as aliposome. Viral vector delivery systems include DNA and RNA viruses,which have either episomal or integrated genomes after delivery to thecell. For a review of gene therapy procedures, see Anderson, Science256:808-813 (1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani &Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993);Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology6(10):1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin51(1):31-44 (1995); Haddada et al., in Current Topics in Microbiologyand Immunology Doerfler and Böhm (eds) (1995); and Yu et al., GeneTherapy 1:13-26 (1994).

Methods of non-viral delivery of nucleic acids include lipofection,microinjection, biolistics, virosomes, liposomes, immunoliposomes,polycation or lipid:nucleic acid conjugates, naked DNA, artificialvirions, and agent-enhanced uptake of DNA. Lipofection is described ine.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) andlipofection reagents are sold commercially (e.g., Transfectam™ andLipofectin™). Cationic and neutral lipids that are suitable forefficient receptor-recognition lipofection of polynucleotides includethose of Felgner, WO 91/17424; WO 91/16024. Delivery can be to cells(e.g., in vitro or ex vivo administration) or target tissues (e.g., invivo administration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

The use of RNA or DNA viral based systems for the delivery of nucleicacids take advantage of highly evolved processes for targeting a virusto specific cells in the body and trafficking the viral payload to thenucleus. Viral vectors can be administered directly to patients (invivo) or they can be used to treat cells in vitro, and the modifiedcells may optionally be administered to patients (ex vivo). Conventionalviral based systems could include retroviral, lentivirus, adenoviral,adeno-associated and herpes simplex virus vectors for gene transfer.Integration in the host genome is possible with the retrovirus,lentivirus, and adeno-associated virus gene transfer methods, oftenresulting in long term expression of the inserted transgene.Additionally, high transduction efficiencies have been observed in manydifferent cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system would thereforedepend on the target tissue. Retroviral vectors are comprised ofcis-acting long terminal repeats with packaging capacity for up to 6-10kb of foreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of the vectors, which are then used tointegrate the therapeutic gene into the target cell to provide permanenttransgene expression. Widely used retroviral vectors include those basedupon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV),Simian Immuno deficiency virus (SIV), human immuno deficiency virus(HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700).

In another embodiment, Cocal vesiculovirus envelope pseudotypedretroviral vector particles are contemplated (see, e.g., US PatentPublication No. 20120164118 assigned to the Fred Hutchinson CancerResearch Center). Cocal virus is in the Vesiculovirus genus, and is acausative agent of vesicular stomatitis in mammals. Cocal virus wasoriginally isolated from mites in Trinidad (Jonkers et al., Am. J Vet.Res. 25:236-242 (1964)), and infections have been identified inTrinidad, Brazil, and Argentina from insects, cattle, and horses. Manyof the vesiculoviruses that infect mammals have been isolated fromnaturally infected arthropods, suggesting that they are vector-bome.Antibodies to vesiculoviruses are common among people living in ruralareas where the viruses are endemic and laboratory-acquired; infectionsin humans usually result in influenza-like symptoms. The Cocal virusenvelope glycoprotein shares 71.5% identity at the amino acid level withVSV-G Indiana, and phylogenetic comparison of the envelope gene ofvesiculoviruses shows that Cocal virus is serologically distinct from,but most closely related to, VSV-G Indiana strains among thevesiculoviruses. Jonkers et al., Am. J. Vet. Res. 25:236-242 (1964) andTravassos da Rosa et al., Am. J. Tropical Med. & Hygiene 33:999-1006(1984). The Cocal vesiculovirus envelope pseudotyped retroviral vectorparticles may include for example, lentiviral, alpharetroviral,betaretroviral, gammaretroviral, deltaretroviral, and epsilonretroviralvector particles that may comprise retroviral Gag, Pol, and/or one ormore accessory protein(s) and a Cocal vesiculovirus envelope protein.Within certain aspects of these embodiments, the Gag, Pol, and accessoryproteins are lentiviral and/or gammaretroviral. The invention providesAAV that contains or consists essentially of an exogenous nucleic acidmolecule encoding a CRISPR system, e.g., a plurality of cassettescomprising or consisting a first cassette comprising or consistingessentially of a promoter, a nucleic acid molecule encoding aCRISPR-associated (Cas) protein (putative nuclease or helicaseproteins), e.g., C2c1 or C2c3 and a terminator, and a two, or more,advantageously up to the packaging size limit of the vector, e.g., intotal (including the first cassette) five, cassettes comprising orconsisting essentially of a promoter, nucleic acid molecule encodingguide RNA (gRNA) and a terminator (e.g., each cassette schematicallyrepresented as Promoter-gRNA1-terminator, Promoter-gRNA2-terminator . .. Promoter-gRNA(N)-terminator (where N is a number that can be insertedthat is at an upper limit of the packaging size limit of the vector), ortwo or more individual rAAVs, each containing one or more than onecassette of a CRISPR system, e.g., a first rAAV containing the firstcassette comprising or consisting essentially of a promoter, a nucleicacid molecule encoding Cas, e.g., Cas (C2c1 or C2c3) and a terminator,and a second rAAV containing a plurality, four, cassettes comprising orconsisting essentially of a promoter, nucleic acid molecule encodingguide RNA (gRNA) and a terminator (e.g., each cassette schematicallyrepresented as Promoter-gRNA1-terminator, Promoter-gRNA2-terminator . .. Promoter-gRNA(N)-terminator (where N is a number that can be insertedthat is at an upper limit of the packaging size limit of the vector). AsrAAV is a DNA virus, the nucleic acid molecules in the herein discussionconcerning AAV or rAAV are advantageously DNA. The promoter is in someembodiments advantageously human Synapsin I promoter (hSyn). Additionalmethods for the delivery of nucleic acids to cells are known to thoseskilled in the art. See, for example, US20030087817, incorporated hereinby reference.

In some embodiments, a host cell is transiently or non-transientlytransfected with one or more vectors described herein. In someembodiments, a cell is transfected as it naturally occurs in a subject.In some embodiments, a cell that is transfected is taken from a subject.In some embodiments, the cell is derived from cells taken from asubject, such as a cell line. A wide variety of cell lines for tissueculture are known in the art. Examples of cell lines include, but arenot limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1,Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1,CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, SW480,SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55,Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E,MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1monkey kidney epithelial, BALB/3T3 mouse embryo fibroblast, 3T3 Swiss,3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T,3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549,ALC, B16, B35, BCP-1 cells, BEAS-2B, bEnd.3, BHK-21, BR 293, BxPC3,C3H-10T1/2, C6/36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T,CHO Dhfr−/−, COR-L23, COR-L23/CPR, COR-L23/5010, COR-L23/R23, COS-7,COV-434, CML T1, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3,EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa,Hepalclc7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812,KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231,MDA-MB-468, MDA-MB-435, MDCK IL, MDCK IL, MOR/0.2R, MONO-MAC 6, MTD-1A,MyEnd, NCI-H69/CPR, NCI-H69/LX10, NCI-H69/LX20, NCI-H69/LX4, NIH-3T3,NALM-1, NW-145, OPCN/OPCT cell lines, Peer, PNT-1A/PNT 2, RenCa, RIN-5F,RMA/RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line,U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, andtransgenic varieties thereof. Cell lines are available from a variety ofsources known to those with skill in the art (see, e.g., the AmericanType Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, acell transfected with one or more vectors described herein is used toestablish a new cell line comprising one or more vector-derivedsequences. In some embodiments, a cell transiently transfected with thecomponents of a nucleic acid-targeting system as described herein (suchas by transient transfection of one or more vectors, or transfectionwith RNA), and modified through the activity of a nucleic acid-targetingcomplex, is used to establish a new cell line comprising cellscontaining the modification but lacking any other exogenous sequence. Insome embodiments, cells transiently or non-transiently transfected withone or more vectors described herein, or cell lines derived from suchcells are used in assessing one or more test compounds.

In some embodiments, one or more vectors described herein are used toproduce a non-human transgenic animal or transgenic plant. In someembodiments, the transgenic animal is a mammal, such as a mouse, rat, orrabbit. In certain embodiments, the organism or subject is a plant. Incertain embodiments, the organism or subject or plant is algae. Methodsfor producing transgenic plants and animals are known in the art, andgenerally begin with a method of cell transfection, such as describedherein. In another embodiment, a fluid delivery device with an array ofneedles (see, e.g., US Patent Publication No. 20110230839 assigned tothe Fred Hutchinson Cancer Research Center) may be contemplated fordelivery of CRISPR Cas to solid tissue. A device of US PatentPublication No. 20110230839 for delivery of a fluid to a solid tissuemay comprise a plurality of needles arranged in an array; a plurality ofreservoirs, each in fluid communication with a respective one of theplurality of needles; and a plurality of actuators operatively coupledto respective ones of the plurality of reservoirs and configured tocontrol a fluid pressure within the reservoir. In certain embodimentseach of the plurality of actuators may comprise one of a plurality ofplungers, a first end of each of the plurality of plungers beingreceived in a respective one of the plurality of reservoirs, and incertain further embodiments the plungers of the plurality of plungersare operatively coupled together at respective second ends so as to besimultaneously depressable. Certain still further embodiments maycomprise a plunger driver configured to depress all of the plurality ofplungers at a selectively variable rate. In other embodiments each ofthe plurality of actuators may comprise one of a plurality of fluidtransmission lines having first and second ends, a first end of each ofthe plurality of fluid transmission lines being coupled to a respectiveone of the plurality of reservoirs. In other embodiments the device maycomprise a fluid pressure source, and each of the plurality of actuatorscomprises a fluid coupling between the fluid pressure source and arespective one of the plurality of reservoirs. In further embodimentsthe fluid pressure source may comprise at least one of a compressor, avacuum accumulator, a peristaltic pump, a master cylinder, amicrofluidic pump, and a valve. In another embodiment, each of theplurality of needles may comprise a plurality of ports distributed alongits length.

In one aspect, the invention provides for methods of modifying a targetpolynucleotide in a eukaryotic cell. In some embodiments, the methodcomprises allowing a nucleic acid-targeting complex to bind to thetarget polynucleotide to effect cleavage of said target polynucleotidethereby modifying the target polynucleotide, wherein the nucleicacid-targeting complex comprises a nucleic acid-targeting effectorprotein complexed with a guide RNA hybridized to a target sequencewithin said target polynucleotide.

In one aspect, the invention provides a method of modifying expressionof a polynucleotide in a eukaryotic cell. In some embodiments, themethod comprises allowing a nucleic acid-targeting complex to bind tothe polynucleotide such that said binding results in increased ordecreased expression of said polynucleotide; wherein the nucleicacid-targeting complex comprises a nucleic acid-targeting effectorprotein complexed with a guide RNA hybridized to a target sequencewithin said polynucleotide.

CRISPR complex components may be delivered by conjugation or associationwith transport moieties (adapted for example from approaches disclosedin U.S. Pat. Nos. 8,106,022; 8,313,772). Nucleic acid deliverystrategies may for example be used to improve delivery of guide RNA, ormessenger RNAs or coding DNAs encoding CRISPR complex components. Forexample, RNAs may incorporate modified RNA nucleotides to improvestability, reduce immunostimulation, and/or improve specificity (seeDeleavey, Glen F. et al., 2012, Chemistry & Biology, Volume 19, Issue 8,937-954; Zalipsky, 1995, Advanced Drug Delivery Reviews 16: 157-182;Caliceti and Veronese, 2003, Advanced Drug Delivery Reviews 55:1261-1277). Various constructs have been described that may be used tomodify nucleic acids, such as gRNAs, for more efficient delivery, suchas reversible charge-neutralizing phosphotriester backbone modificationsthat may be adapted to modify gRNAs so as to be more hydrophobic andnon-anionic, thereby improving cell entry (Meade B R et al., 2014,Nature Biotechnology 32, 1256-1261). In further alternative embodiments,selected RNA motifs may be useful for mediating cellular transfection(Magalhies M., et al., Molecular Therapy (2012); 20 3, 616-624).Similarly, aptamers may be adapted for delivery of CRISPR complexcomponents, for example by appending aptamers to gRNAs (Tan W. et al.,2011, Trends in Biotechnology, December 2011, Vol. 29, No. 12).

In some embodiments, conjugation of triantennary N-acetyl galactosamine(GalNAc) to oligonucleotide components may be used to improve delivery,for example delivery to select cell types, for example hepatocytes (seeWO2014118272 incorporated herein by reference; Nair, J K et al., 2014,Journal of the American Chemical Society 136 (49), 16958-16961). Thismay be is considered to be a sugar-based particle and further details onother particle delivery systems and/or formulations are provided herein.GalNAc can therefore be considered to be a particle in the sense of theother particles described herein, such that general uses and otherconsiderations, for instance delivery of said particles, apply to GalNAcparticles as well. A solution-phase conjugation strategy may for examplebe used to attach triantennary GalNAc clusters (mol. wt. ˜2000)activated as PFP (pentafluorophenyl) esters onto 5′-hexylamino modifiedoligonucleotides (5′-HA ASOs, mol. wt. −8000 Da; Ostergaard et al.,Bioconjugate Chem., 2015, 26 (8), pp 1451-1455). Similarly,poly(acrylate) polymers have been described for in vivo nucleic aciddelivery (see WO2013158141 incorporated herein by reference). In furtheralternative embodiments, pre-mixing CRISPR nanoparticles (or proteincomplexes) with naturally occurring serum proteins may be used in orderto improve delivery (Akinc A et al, 2010, Molecular Therapy vol. 18 no.7, 1357-1364).

Screening techniques are available to identify delivery enhancers, forexample by screening chemical libraries (Gilleron J. et al., 2015, Nucl.Acids Res. 43 (16): 7984-8001). Approaches have also been described forassessing the efficiency of delivery vehicles, such as lipidnanoparticles, which may be employed to identify effective deliveryvehicles for CRISPR components (see Sahay G. et al., 2013, NatureBiotechnology 31, 653-658).

In some embodiments, delivery of protein CRISPR components may befacilitated with the addition of functional peptides to the protein,such as peptides that change protein hydrophobicity, for example so asto improve in vivo functionality. CRISPR component proteins maysimilarly be modified to facilitate subsequent chemical reactions. Forexample, amino acids may be added to a protein that have a group thatundergoes click chemistry (Nikié I. et al., 2015, Nature Protocols 10,780-791). In embodiments of this kind, the click chemical group may thenbe used to add a wide variety of alternative structures, such aspoly(ethylene glycol) for stability, cell penetrating peptides, RNAaptamers, lipids, or carbohydrates such as GalNAc. In furtheralternatives, a CRISPR component protein may be modified to adapt theprotein for cell entry (see Svensen et al., 2012, Trends inPharmacological Sciences, Vol. 33, No. 4), for example by adding cellpenetrating peptides to the protein (see Kauffman, W. Berkeley et al.,2015, Trends in Biochemical Sciences, Volume 40, Issue 12, 749-764;Koren and Torchilin, 2012, Trends in Molecular Medicine, Vol. 18, No.7). In further alternative embodiment, patients or subjects may bepre-treated with compounds or formulations that facilitate the laterdelivery of CRISPR components.

C2c1 or C2c3Effector Protein Complexes can be Used in Plants

The C2c1 or C2c3 effector protein system(s) (e.g., single ormultiplexed) can be used in conjunction with recent advances in cropgenomics. The systems described herein can be used to perform efficientand cost effective plant gene or genome interrogation or editing ormanipulation—for instance, for rapid investigation and/or selectionand/or interrogations and/or comparison and/or manipulations and/ortransformation of plant genes or genomes; e.g., to create, identify,develop, optimize, or confer trait(s) or characteristic(s) to plant(s)or to transform a plant genome. There can accordingly be improvedproduction of plants, new plants with new combinations of traits orcharacteristics or new plants with enhanced traits. The C2c1 or C2c3effector protein system(s) can be used with regard to plants inSite-Directed Integration (SDI) or Gene Editing (GE) or any Near ReverseBreeding (NRB) or Reverse Breeding (RB) techniques. Aspects of utilizingthe herein described C2c1 or C2c3 effector protein systems may beanalogous to the use of the CRISPR-Cas (e.g. CRISPR-Cas9) system inplants, and mention is made of the University of Arizona website“CRISPR-PLANT” (www.genome.arizona.edu/crispr/) (supported by Penn Stateand AGI). Embodiments of the invention can be used in genome editing inplants or where RNAi or similar genome editing techniques have been usedpreviously; see, e.g., Nekrasov, “Plant genome editing made easy:targeted mutagenesis in model and crop plants using the CRISPR-Cassystem,” Plant Methods 2013, 9:39 (doi:10.1186/1746-4811-9-39); Brooks,“Efficient gene editing in tomato in the first generation using theCRISPR-Cas9 system,” Plant Physiology September 2014 pp 114.247577;Shan, “Targeted genome modification of crop plants using a CRISPR-Cassystem,” Nature Biotechnology 31, 686-688 (2013); Feng, “Efficientgenome editing in plants using a CRISPR/Cas system,” Cell Research(2013) 23:1229-1232. doi:10.1038/cr.2013.114; published online 20 Aug.2013; Xie, “RNA-guided genome editing in plants using a CRISPR-Cassystem,” Mol Plant. 2013 November; 6(6):1975-83. doi: 10.1093/mp/sst119.Epub 2013 Aug. 17; Xu, “Gene targeting using the Agrobacteriumtumefaciens-mediated CRISPR-Cas system in rice,” Rice 2014, 7:5 (2014),Zhou et al., “Exploiting SNPs for biallelic CRISPR mutations in theoutcrossing woody perennial Populus reveals 4-coumarate: CoA ligasespecificity and Redundancy,” New Phytologist (2015) (Forum) 1-4(available online only at www.newphytologist.com); Caliando et al,“Targeted DNA degradation using a CRISPR device stably carried in thehost genome, NATURE COMMUNICATIONS 6:6989, DOI: 10.1038/ncomms7989,www.nature.com/naturecommunications DOI: 10.1038/ncomms7989; U.S. Pat.No. 6,603,061—Agrobacterium-Mediated Plant Transformation Method; U.S.Pat. No. 7,868,149—Plant Genome Sequences and Uses Thereof and US2009/0100536—Transgenic Plants with Enhanced Agronomic Traits, all thecontents and disclosure of each of which are herein incorporated byreference in their entirety. In the practice of the invention, thecontents and disclosure of Morrell et al “Crop genomics: advances andapplications,” Nat Rev Genet. 2011 Dec. 29; 13(2):85-96; each of whichis incorporated by reference herein including as to how hereinembodiments may be used as to plants. Accordingly, reference herein toanimal cells may also apply, mutatis mutandis, to plant cells unlessotherwise apparent; and, the enzymes herein having reduced off-targeteffects and systems employing such enzymes can be used in plantapplications, including those mentioned herein.

Application of C2c1- or C2c3-CRISPR System to Plants and Yeast

Definition

In general, the term “plant” relates to any various photosynthetic,eukaryotic, unicellular or multicellular organism of the kingdom Plantaecharacteristically growing by cell division, containing chloroplasts,and having cell walls comprised of cellulose. The term plant encompassesmonocotyledonous and dicotyledonous plants. Specifically, the plants areintended to comprise without limitation angiosperm and gymnosperm plantssuch as acacia, alfalfa, amaranth, apple, apricot, artichoke, ash tree,asparagus, avocado, banana, barley, beans, beet, birch, beech,blackberry, blueberry, broccoli, Brussel's sprouts, cabbage, canola,cantaloupe, carrot, cassava, cauliflower, cedar, a cereal, celery,chestnut, cherry, Chinese cabbage, citrus, clementine, clover, coffee,corn, cotton, cowpea, cucumber, cypress, eggplant, elm, endive,eucalyptus, fennel, figs, fir, geranium, grape, grapefruit, groundnuts,ground cherry, gum hemlock, hickory, kale, kiwifruit, kohlrabi, larch,lettuce, leek, lemon, lime, locust, pine, maidenhair, maize, mango,maple, melon, millet, mushroom, mustard, nuts, oak, oats, oil palm,okra, onion, orange, an ornamental plant or flower or tree, papaya,palm, parsley, parsnip, pea, peach, peanut, pear, peat, pepper,persimmon, pigeon pea, pine, pineapple, plantain, plum, pomegranate,potato, pumpkin, radicchio, radish, rapeseed, raspberry, rice, rye,sorghum, safflower, sallow, soybean, spinach, spruce, squash,strawberry, sugar beet, sugarcane, sunflower, sweet potato, sweet corn,tangerine, tea, tobacco, tomato, trees, triticale, turf grasses,turnips, vine, walnut, watercress, watermelon, wheat, yams, yew, andzucchini. The term plant also encompasses Algae, which are mainlyphotoautotrophs unified primarily by their lack of roots, leaves andother organs that characterize higher plants.

The methods for genome editing using the C2c1 or C2c3 system asdescribed herein can be used to confer desired traits on essentially anyplant. A wide variety of plants and plant cell systems may be engineeredfor the desired physiological and agronomic characteristics describedherein using the nucleic acid constructs of the present disclosure andthe various transformation methods mentioned above. In preferredembodiments, target plants and plant cells for engineering include, butare not limited to, those monocotyledonous and dicotyledonous plants,such as crops including grain crops (e.g., wheat, maize, rice, millet,barley), fruit crops (e.g., tomato, apple, pear, strawberry, orange),forage crops (e.g., alfalfa), root vegetable crops (e.g., carrot,potato, sugar beets, yam), leafy vegetable crops (e.g., lettuce,spinach); flowering plants (e.g., petunia, rose, chrysanthemum),conifers and pine trees (e.g., pine fir, spruce); plants used inphytoremediation (e.g., heavy metal accumulating plants); oil crops(e.g., sunflower, rape seed) and plants used for experimental purposes(e.g., Arabidopsis). Thus, the methods and CRISPR-Cas systems can beused over a broad range of plants, such as for example withdicotyledonous plants belonging to the orders Magnoliales, Illiciales,Laurales, Piperales, Aristolochiales, Nymphaeales, Ranunculales,Papeverales, Sarraceniaceae, Trochodendrales, Hamamelidales, Eucomiales,Leitneriales, Myricales, Fagales, Casuarinales, Caryophyllales, Batales,Polygonales, Plumbaginales, Dilleniales, Theales, Malvales, Urticales,Lecythidales, Violales, Salicales, Capparales, Ericales, Diapensales,Ebenales, Primulales, Rosales, Fabales, Podostemales, Haloragales,Myrtales, Cornales, Proteales, San tales, Rafflesiales, Celastrales,Euphorbiales, Rhamnales, Sapindales, Juglandales, Geraniales,Polygalales, Umbellales, Gentianales, Polemoniales, Lamiales,Plantaginales, Scrophulariales, Campanulales, Rubiales, Dipsacales, andAsterales; the methods and CRISPR-Cas systems can be used withmonocotyledonous plants such as those belonging to the ordersAlismatales, Hydrocharitales, Najadales, Triuridales, Commelinales,Eriocaulales, Restionales, Poales, Juncales, Cyperales, Typhales,Bromeliales, Zingiberales, Arecales, Cyclanthales, Pandanales, Arales,Liliales, and Orchid ales, or with plants belonging to Gymnospermae, e.gthose belonging to the orders Pinales, Ginkgoales, Cycadales,Araucariales, Cupressales and Gnetales.

The C2c1 or C2c3 CRISPR systems and methods of use described herein canbe used over a broad range of plant species, included in thenon-limitative list of dicot, monocot or gymnosperm genera hereunder:Atropa, Alseodaphne, Anacardium, Arachis, Beilschmiedia, Brassica,Carthamus, Cocculus, Croton, Cucumis, Citrus, Citrullus, Capsicum,Catharanthus, Cocos, Cofea, Cucurbita, Daucus, Duguetia, Eschscholzia,Ficus, Fragaria, Glaucium, Glycine, Gossypium, Helianthus, Hevea,Hyoscyamus, Lactuca, Landolphia, Linum, Litsea, Lycopersicon, Lupimis,Manihot, Majorana, Malus, Medicago. Nicotiana. Olea, Parthenium,Papaver. Persea, Phaseolus, Pistacia, Pisum, Pyrus, Prunus, Raphanus,Ricinus, Senecio, Sinomenium, Stephania, Sinapis, Solanum, Theobroma,Trifolium, Trigonella, Vicia, Vinca, Vilis, and Vigna; and the generaAllium, Andropogon, Eragrostis, Asparagus, Avena, Cynodon, Elaeis,Festuca, Festulolium, Heterocallis, Hordeum, Lemna, Lolium, Musa, Oryza,Panicum, Pennisetum, Phleum, Poa, Secale, Sorghum, Triticum, Zea, Abies,Cunninghamia, Ephedra, Picea, Pinus, and Pseudotsuga.

The C2c1 or C2c3 CRISPR systems and methods of use can also be used overa broad range of “algae” or “algae cells”; including for example algaeselected from several eukaryotic phyla, including the Rhodophyta (redalgae), Chlorophyta (green algae), Phaeophyta (brown algae),Bacillariophyta (diatoms), Eustigmatophyta and dinoflagellates as wellas the prokaryotic phylum Cyanobacteria (blue-green algae). The term“algae” includes for example algae selected from: Amphora, Anabaena,Ankistrodesmus, Botryococcus, Chaetoceros, Chlamydomonas, Chlorella,Chlorococcum, Cyclotella, Cylindrotheca, Dunaliella, Emiliana, Euglena,Haematococcus, Isochrysis, Monochrysis, Monoraphidium, Nannochloris,Nannochloropsis, Navicula, Nephrochloris, Nephroselmis, Nitzschia,Nodularia, Nostoc, Ochromonas, Oocystis, Oscillatoria, Pavlova,Phaeodactylum, Platymonas, Pleurochrysis, Porhyra, Pseudoanabaena,Pyramimonas, Stichococcus, Synechococcus, Synechocystis, Tetraselmis,Thalassiosira, and Trichodesmium.

A part of a plant, i.e., a “plant tissue” may be treated according tothe methods of the present invention to produce an improved plant. Planttissue also encompasses plant cells. The term “plant cell” as usedherein refers to individual units of a living plant, either in an intactwhole plant or in an isolated form grown in in vitro tissue cultures, onmedia or agar, in suspension in a growth media or buffer or as a part ofhigher organized unites, such as, for example, plant tissue, a plantorgan, or a whole plant.

A “protoplast” refers to a plant cell that has had its protective cellwall completely or partially removed using, for example, mechanical orenzymatic means resulting in an intact biochemical competent unit ofliving plant that can reform their cell wall, proliferate and regenerategrow into a whole plant under proper growing conditions.

The term “transformation” broadly refers to the process by which a planthost is genetically modified by the introduction of DNA by means ofAgrobacteria or one of a variety of chemical or physical methods. Asused herein, the term “plant host” refers to plants, including anycells, tissues, organs, or progeny of the plants. Many suitable planttissues or plant cells can be transformed and include, but are notlimited to, protoplasts, somatic embryos, pollen, leaves, seedlings,stems, calli, stolons, microtubers, and shoots. A plant tissue alsorefers to any clone of such a plant, seed, progeny, propagule whethergenerated sexually or asexually, and descendents of any of these, suchas cuttings or seed.

The term “transformed” as used herein, refers to a cell, tissue, organ,or organism into which a foreign DNA molecule, such as a construct, hasbeen introduced. The introduced DNA molecule may be integrated into thegenomic DNA of the recipient cell, tissue, organ, or organism such thatthe introduced DNA molecule is transmitted to the subsequent progeny. Inthese embodiments, the “transformed” or “transgenic” cell or plant mayalso include progeny of the cell or plant and progeny produced from abreeding program employing such a transformed plant as a parent in across and exhibiting an altered phenotype resulting from the presence ofthe introduced DNA molecule. Preferably, the transgenic plant is fertileand capable of transmitting the introduced DNA to progeny through sexualreproduction.

The term “progeny”, such as the progeny of a transgenic plant, is onethat is born of, begotten by, or derived from a plant or the transgenicplant. The introduced DNA molecule may also be transiently introducedinto the recipient cell such that the introduced DNA molecule is notinherited by subsequent progeny and thus not considered “transgenic”.Accordingly, as used herein, a “non-transgenic” plant or plant cell is aplant which does not contain a foreign DNA stably integrated into itsgenome.

The term “plant promoter” as used herein is a promoter capable ofinitiating transcription in plant cells, whether or not its origin is aplant cell. Exemplary suitable plant promoters include, but are notlimited to, those that are obtained from plants, plant viruses, andbacteria such as Agrobacterium or Rhizobium which comprise genesexpressed in plant cells.

As used herein, a “fungal cell” refers to any type of eukaryotic cellwithin the kingdom of fungi. Phyla within the kingdom of fungi includeAscomycota, Basidiomycota, Blastocladiomycota, Chytridiomycota,Glomeromycota, Microsporidia, and Neocallimastigomycota. Fungal cellsmay include yeasts, molds, and filamentous fungi. In some embodiments,the fungal cell is a yeast cell.

As used herein, the term “yeast cell” refers to any fungal cell withinthe phyla Ascomycota and Basidiomycota. Yeast cells may include buddingyeast cells, fission yeast cells, and mold cells. Without being limitedto these organisms, many types of yeast used in laboratory andindustrial settings are part of the phylum Ascomycota. In someembodiments, the yeast cell is an S. cerevisiae, Kluyveromycesmarxianus, or Issatchenkia orientalis cell. Other yeast cells mayinclude without limitation Candida spp. (e.g., Candida albicans),Yarrowia spp. (e.g., Yarrowia lipolytica), Pichia spp. (e.g., Pichiapastoris), Kluyveromyces spp. (e.g., Kluyveromyces lactis andKluyveromyces marxianus), Neurospora spp. (e.g., Neurospora crassa),Fusarium spp. (e.g., Fusarium oxysporum), and Issatchenkia spp. (e.g.,Issatchenkia orientalis, a.k.a. Pichia kudriavzevii and Candidaacidothermophilum). In some embodiments, the fungal cell is afilamentous fungal cell. As used herein, the term “filamentous fungalcell” refers to any type of fungal cell that grows in filaments, i.e.,hyphae or mycelia. Examples of filamentous fungal cells may includewithout limitation Aspergillus spp. (e.g., Aspergillus niger),Trichoderma spp. (e.g., Trichoderma reesei), Rhizopus spp. (e.g.,Rhizopus oryzae), and Mortierella spp. (e.g., Mortierella isabellina).

In some embodiments, the fungal cell is an industrial strain. As usedherein, “industrial strain” refers to any strain of fungal cell used inor isolated from an industrial process, e.g., production of a product ona commercial or industrial scale. Industrial strain may refer to afungal species that is typically used in an industrial process, or itmay refer to an isolate of a fungal species that may be also used fornon-industrial purposes (e.g., laboratory research). Examples ofindustrial processes may include fermentation (e.g., in production offood or beverage products), distillation, biofuel production, productionof a compound, and production of a polypeptide. Examples of industrialstrains may include, without limitation, JAY270 and ATCC4124.

In some embodiments, the fungal cell is a polyploid cell. As usedherein, a “polyploid” cell may refer to any cell whose genome is presentin more than one copy. A polyploid cell may refer to a type of cell thatis naturally found in a polyploid state, or it may refer to a cell thathas been induced to exist in a polyploid state (e.g., through specificregulation, alteration, inactivation, activation, or modification ofmeiosis, cytokinesis, or DNA replication). A polyploid cell may refer toa cell whose entire genome is polyploid, or it may refer to a cell thatis polyploid in a particular genomic locus of interest. Without wishingto be bound to theory, it is thought that the abundance of guideRNA maymore often be a rate-limiting component in genome engineering ofpolyploid cells than in haploid cells, and thus the methods using theC2c1 or C2c3 CRISPRS system described herein may take advantage of usinga certain fungal cell type.

In some embodiments, the fungal cell is a diploid cell. As used herein,a “diploid” cell may refer to any cell whose genome is present in twocopies. A diploid cell may refer to a type of cell that is naturallyfound in a diploid state, or it may refer to a cell that has beeninduced to exist in a diploid state (e.g., through specific regulation,alteration, inactivation, activation, or modification of meiosis,cytokinesis, or DNA replication). For example, the S. cerevisiae strainS228C may be maintained in a haploid or diploid state. A diploid cellmay refer to a cell whose entire genome is diploid, or it may refer to acell that is diploid in a particular genomic locus of interest. In someembodiments, the fungal cell is a haploid cell. As used herein, a“haploid” cell may refer to any cell whose genome is present in onecopy. A haploid cell may refer to a type of cell that is naturally foundin a haploid state, or it may refer to a cell that has been induced toexist in a haploid state (e.g., through specific regulation, alteration,inactivation, activation, or modification of meiosis, cytokinesis, orDNA replication). For example, the S. cerevisiae strain S228C may bemaintained in a haploid or diploid state. A haploid cell may refer to acell whose entire genome is haploid, or it may refer to a cell that ishaploid in a particular genomic locus of interest.

As used herein, a “yeast expression vector” refers to a nucleic acidthat contains one or more sequences encoding an RNA and/or polypeptideand may further contain any desired elements that control the expressionof the nucleic acid(s), as well as any elements that enable thereplication and maintenance of the expression vector inside the yeastcell. Many suitable yeast expression vectors and features thereof areknown in the art; for example, various vectors and techniques areillustrated in in Yeast Protocols, 2nd edition, Xiao, W., ed. (HumanaPress, New York, 2007) and Buckholz, R. G. and Gleeson, M. A. (1991)Biotechnology (NY) 9(11): 1067-72. Yeast vectors may contain, withoutlimitation, a centromeric (CEN) sequence, an autonomous replicationsequence (ARS), a promoter, such as an RNA Polymerase III promoter,operably linked to a sequence or gene of interest, a terminator such asan RNA polymerase III terminator, an origin of replication, and a markergene (e.g., auxotrophic, antibiotic, or other selectable markers).Examples of expression vectors for use in yeast may include plasmids,yeast artificial chromosomes, 2μ plasmids, yeast integrative plasmids,yeast replicative plasmids, shuttle vectors, and episomal plasmids.

Stable Integration of C2c1 or C2e3 CRISPR System Components in theGenome of Plants and Plant Cells

In particular embodiments, it is envisaged that the polynucleotidesencoding the components of the C2c1 or C2c3 CRISPR system are introducedfor stable integration into the genome of a plant cell. In theseembodiments, the design of the transformation vector or the expressionsystem can be adjusted depending on for when, where and under whatconditions the guide RNA and/or the C2c1 or C2c3 gene are expressed.

In particular embodiments, it is envisaged to introduce the componentsof the C2c1 or C2c3 CRISPR system stably into the genomic DNA of a plantcell. Additionally or alternatively, it is envisaged to introduce thecomponents of the C2c1 or C2c3 CRISPR system for stable integration intothe DNA of a plant organelle such as, but not limited to a plastid, amitochondrion or a chloroplast.

The expression system for stable integration into the genome of a plantcell may contain one or more of the following elements: a promoterelement that can be used to express the RNA and/or C2c1 or C2c3 enzymein a plant cell; a 5′ untranslated region to enhance expression; anintron element to further enhance expression in certain cells, such asmonocot cells; a multiple-cloning site to provide convenient restrictionsites for inserting the guide RNA and/or the C2c1 or C2c3 gene sequencesand other desired elements; and a 3′ untranslated region to provide forefficient termination of the expressed transcript.

The elements of the expression system may be on one or more expressionconstructs which are either circular such as a plasmid or transformationvector, or non-circular such as linear double stranded DNA.

In a particular embodiment, a C2c1 or C2c3 CRISPR expression systemcomprises at least:

-   (a) a nucleotide sequence encoding a guide RNA (gRNA) that    hybridizes with a target sequence in a plant, and wherein the guide    RNA comprises a guide sequence and a direct repeat sequence, and-   (b) a nucleotide sequence encoding a C2c1 or C2c3 protein,

wherein components (a) or (b) are located on the same or on differentconstructs, and whereby the different nucleotide sequences can be undercontrol of the same or a different regulatory element operable in aplant cell.

DNA construct(s) containing the components of the C2c1 or C2c3 CRISPRsystem, and, where applicable, template sequence may be introduced intothe genome of a plant, plant part, or plant cell by a variety ofconventional techniques. The process generally comprises the steps ofselecting a suitable host cell or host tissue, introducing theconstruct(s) into the host cell or host tissue, and regenerating plantcells or plants therefrom.

In particular embodiments, the DNA construct may be introduced into theplant cell using techniques such as but not limited to electroporation,microinjection, aerosol beam injection of plant cell protoplasts, or theDNA constructs can be introduced directly to plant tissue usingbiolistic methods, such as DNA particle bombardment (see also Fu et al.,Transgenic Res. 2000 February; 9(1):11-9). The basis of particlebombardment is the acceleration of particles coated with gene/s ofinterest toward cells, resulting in the penetration of the protoplasm bythe particles and typically stable integration into the genome. (seee.g. Klein et al., Nature (1987), Klein et al., Bio/Technology (1992),Casas et al., Proc. Natl. Acad Sci. USA (1993).).

In particular embodiments, the DNA constructs containing components ofthe C2c1 or C2c3 CRISPR system may be introduced into the plant byAgrobacterium-mediated transformation. The DNA constructs may becombined with suitable T-DNA flanking regions and introduced into aconventional Agrobacterium tumefaciens host vector. The foreign DNA canbe incorporated into the genome of plants by infecting the plants or byincubating plant protoplasts with Agrobacterium bacteria, containing oneor more Ti (tumor-inducing) plasmids. (see e.g. Fraley et al., (1985),Rogers et al., (1987) and U.S. Pat. No. 5,563,055).

Plant Promoters

In order to ensure appropriate expression in a plant cell, thecomponents of the C2c1 or C2c3 CRISPR system described herein aretypically placed under control of a plant promoter, i.e. a promoteroperable in plant cells. The use of different types of promoters isenvisaged.

A constitutive plant promoter is a promoter that is able to express theopen reading frame (ORF) that it controls in all or nearly all of theplant tissues during all or nearly all developmental stages of the plant(referred to as “constitutive expression”). One non-limiting example ofa constitutive promoter is the cauliflower mosaic virus 35S promoter.“Regulated promoter” refers to promoters that direct gene expression notconstitutively, but in a temporally- and/or spatially-regulated manner,and includes tissue-specific, tissue-preferred and inducible promoters.Different promoters may direct the expression of a gene in differenttissues or cell types, or at different stages of development, or inresponse to different environmental conditions. In particularembodiments, one or more of the C2c1 or C2c3 CRISPR components areexpressed under the control of a constitutive promoter, such as thecauliflower mosaic virus 35S promoter issue-preferred promoters can beutilized to target enhanced expression in certain cell types within aparticular plant tissue, for instance vascular cells in leaves or rootsor in specific cells of the seed. Examples of particular promoters foruse in the C2c1 or C2c3 CRISPR system—are found in Kawamata et al.,(1997) Plant Cell Physiol. 38:792-803; Yamamoto et al., (1997) Plant J.12:255-65; Hire et al, (1992) Plant Mol. Biol. 20:207-18, Kuster et al,(1995) Plant Mol. Biol. 29:759-72, and Capana et al., (1994) Plant Mol.Biol. 25:681-91.

Examples of promoters that are inducible and that allow forspatiotemporal control of gene editing or gene expression may use a formof energy. The form of energy may include but is not limited to soundenergy, electromagnetic radiation, chemical energy and/or thermalenergy. Examples of inducible systems include tetracycline induciblepromoters (Tet-On or Tet-Off), small molecule two-hybrid transcriptionactivations systems (FKBP, ABA, etc), or light inducible systems(Phytochrome, LOV domains, or cryptochrome)., such as a Light InducibleTranscriptional Effector (LITE) that direct changes in transcriptionalactivity in a sequence-specific manner. The components of a lightinducible system may include a C2c1 or C2c3 CRISPR enzyme, alight-responsive cytochrome heterodimer (e.g. from Arabidopsisthaliana), and a transcriptional activation/repression domain. Furtherexamples of inducible DNA binding proteins and methods for their use areprovided in U.S. 61/736,465 and U.S. 61/721,283, which is herebyincorporated by reference in its entirety.

In particular embodiments, transient or inducible expression can beachieved by using, for example, chemical-regulated promotors, i.e.whereby the application of an exogenous chemical induces geneexpression. Modulating of gene expression can also be obtained by achemical-repressible promoter, where application of the chemicalrepresses gene expression. Chemical-inducible promoters include, but arenot limited to, the maize ln2-2 promoter, activated by benzenesulfonamide herbicide safeners (De Veylder et al., (1997) Plant CellPhysiol. 38:568-77), the maize GST promoter (GST-ll-27, WO93/01294),activated by hydrophobic electrophilic compounds used as pre-emergentherbicides, and the tobacco PR-1 a promoter (Ono et al., (2004) Biosci.Biotechnol. Biochem. 68:803-7) activated by salicylic acid. Promoterswhich are regulated by antibiotics, such as tetracycline-inducible andtetracycline-repressible promoters (Gatz et al., (1991) Mol. Gen. Genet.227:229-37; U.S. Pat. Nos. 5,814,618 and 5,789,156) can also be usedherein.

Translocation to and/or Expression in Specific Plant Organelles

The expression system may comprise elements for translocation to and/orexpression in a specific plant organelle.

Chloroplast Targeting

In particular embodiments, it is envisaged that the C2c1 or C2c3 CRISPRsystem is used to specifically modify chloroplast genes or to ensureexpression in the chloroplast. For this purpose use is made ofchloroplast transformation methods or compartmentalization of the C2c1or C2c3 CRISPR components to the chloroplast. For instance, theintroduction of genetic modifications in the plastid genome can reducebiosafety issues such as gene flow through pollen.

Methods of chloroplast transformation are known in the art and includeParticle bombardment, PEG treatment, and microinjection. Additionally,methods involving the translocation of transformation cassettes from thenuclear genome to the plastid can be used as described in WO2010061186.

Alternatively, it is envisaged to target one or more of the C2c1 or C2c3CRISPR components to the plant chloroplast. This is achieved byincorporating in the expression construct a sequence encoding achloroplast transit peptide (CTP) or plastid transit peptide, operablylinked to the 5′ region of the sequence encoding the C2c1 or C2c3protein. The CTP is removed in a processing step during translocationinto the chloroplast. Chloroplast targeting of expressed proteins iswell known to the skilled artisan (see for instance Protein Transportinto Chloroplasts, 2010, Annual Review of Plant Biology, Vol. 61:157-180). In such embodiments it is also desired to target the guide RNAto the plant chloroplast. Methods and constructs which can be used fortranslocating guide RNA into the chloroplast by means of a chloroplastlocalization sequence are described, for instance, in US 20040142476,incorporated herein by reference. Such variations of constructs can beincorporated into the expression systems of the invention to efficientlytranslocate the C2c1- or C2c3-guide RNA.

Introduction of Polynucleotides Encoding the CRISPR-C2c1 or CRISPR-C2c3System in Algal Cells

Transgenic algae (or other plants such as rape) may be particularlyuseful in the production of vegetable oils or biofuels such as alcohols(especially methanol and ethanol) or other products. These may beengineered to express or overexpress high levels of oil or alcohols foruse in the oil or biofuel industries.

U.S. Pat. No. 8,945,839 describes a method for engineering Micro-Algae(Chlamydomonas reinhardtii cells) species) using Cas9. Using similartools, the methods of the C2c1 or C2c3 CRISPR system described hereincan be applied on Chlamydomonas species and other algae. In particularembodiments, C2c1 or C2c3 and guide RNA are introduced in algaeexpressed using a vector that expresses C2c1 or C2c3 under the controlof a constitutive promoter such as Hsp70A-Rbc S2 or Beta2-tubulin. GuideRNA is optionally delivered using a vector containing T7 promoter.Alternatively, Cas9 mRNA and in vitro transcribed guide RNA can bedelivered to algal cells. Electroporation protocols are available to theskilled person such as the standard recommended protocol from theGeneArt Chlamydomonas Engineering kit.

In particular embodiments, the endonuclease used herein is a Split C2c1or C2c3 enzyme. Split C2c1 or C2c3 enzymes are preferentially used inAlgae for targeted genome modification as has been described for Cas9 inWO 2015086795. Use of the C2c1 or C2c3 split system is particularlysuitable for an inducible method of genome targeting and avoids thepotential toxic effect of the C2c1 or C2c3 overexpression within thealgae cell. In particular embodiments, Said C2c1 or C2c3 split domains(RuvC and HNH domains) can be simultaneously or sequentially introducedinto the cell such that said split C2c1 or C2c3 domain(s) process thetarget nucleic acid sequence in the algae cell. The reduced size of thesplit C2c1 or C2c3 compared to the wild type C2c1 or C2c3 allows othermethods of delivery of the CRISPR system to the cells, such as the useof Cell Penetrating Peptides as described herein. This method is ofparticular interest for generating genetically modified algae.

Introduction of polynucleotides encoding C2c1 or C2c3 components inyeast cells

In particular embodiments, the invention relates to the use of the C2c1or C2c3 CRISPR system for genome editing of yeast cells. Methods fortransforming yeast cells which can be used to introduce polynucleotidesencoding the C2c1 or C2c3 CRISPR system components are well known to theartisan and are reviewed by Kawai et al., 2010, Bioeng Bugs. 2010November-December; 1(6): 395-403). Non-limiting examples includetransformation of yeast cells by lithium acetate treatment (which mayfurther include carrier DNA and PEG treatment), bombardment or byelectroporation.

Transient Expression of C2c1 or C2e3 CRISPR System Components in Plantsand Plant Cell

In particular embodiments, it is envisaged that the guide RNA and/orC2c1 or C2c3 gene are transiently expressed in the plant cell. In theseembodiments, the C2c1 or C2c3 CRISPR system can ensure modification of atarget gene only when both the guide RNA and the C2c1 or C2c3 protein ispresent in a cell, such that genomic modification can further becontrolled. As the expression of the C2c1 or C2c3 enzyme is transient,plants regenerated from such plant cells typically contain no foreignDNA. In particular embodiments the C2c1 or C2c3 enzyme is stablyexpressed by the plant cell and the guide sequence is transientlyexpressed.

In particular embodiments, the C2c1 or C2c3 CRISPR system components canbe introduced in the plant cells using a plant viral vector (Scholthofet al. 1996, Annu Rev Phywopathol. 1996; 34:299-323). In furtherparticular embodiments, said viral vector is a vector from a DNA virus.For example, geminivirus (e.g., cabbage leaf curl virus, bean yellowdwarf virus, wheat dwarf virus, tomato leaf curl virus, maize streakvirus, tobacco leaf curl virus, or tomato golden mosaic virus) ornanovirus (e.g., Faba bean necrotic yellow virus). In other particularembodiments, said viral vector is a vector from an RNA virus. Forexample, tobravirus (e.g., tobacco rattle virus, tobacco mosaic virus),potexvirus (e.g., potato virus X), or hordeivirus (e.g., barley stripemosaic virus). The replicating genomes of plant viruses arenon-integrative vectors.

In particular embodiments, the vector used for transient expression ofC2c1 or C2c3 CRISPR constructs is for instance a pEAQ vector, which istailored for Agrobacterium-mediated transient expression (Sainsbury F.et al., Plant Biotechnol J. 2009 September; 7(7):682-93) in theprotoplast. Precise targeting of genomic locations was demonstratedusing a modified Cabbage Leaf Curl virus (CaLCuV) vector to expressgRNAs in stable transgenic plants expressing a CRISPR enzyme (ScientificReports 5, Article number: 14926 (2015), doi:10.1038/srep14926).

In particular embodiments, double-stranded DNA fragments encoding theguide RNA and/or the C2c1 or C2c3 gene can be transiently introducedinto the plant cell. In such embodiments, the introduced double-strandedDNA fragments are provided in sufficient quantity to modify the cell butdo not persist after a contemplated period of time has passed or afterone or more cell divisions. Methods for direct DNA transfer in plantsare known by the skilled artisan (see for instance Davey et al., PlantMol. Biol. 1989 September; 13(3):273-85.)

In other embodiments, an RNA polynucleotide encoding the C2c1 or C2c3protein is introduced into the plant cell, which is then translated andprocessed by the host cell generating the protein in sufficient quantityto modify the cell (in the presence of at least one guide RNA) but whichdoes not persist after a contemplated period of time has passed or afterone or more cell divisions. Methods for introducing mRNA to plantprotoplasts for transient expression are known by the skilled artisan(see, for instance in Gallie, Plant Cell Reports (1993), 13; 119-122).Combinations of the different methods described above are alsoenvisaged.

Delivery of C2c1 or C2e3 CRISPR Components to the Plant Cell

In particular embodiments, it is of interest to deliver one or morecomponents of the C2c1 or C2c3 CRISPR system directly to the plant cell.This is of interest, inter alia, for the generation of non-transgenicplants (see below). In particular embodiments, one or more of the C2c1or C2c3 components is prepared outside the plant or plant cell anddelivered to the cell. For instance in particular embodiments, the C2c1or C2c3 protein is prepared in vitro prior to introduction to the plantcell. C2c1 or C2c3 protein can be prepared by various methods known byone of skill in the art and include recombinant production. Afterexpression, the C2c1 or C2c3 protein is isolated, refolded if needed,purified and optionally treated to remove any purification tags, such asa His-tag. Once crude, partially purified, or more completely purifiedC2c1 or C2c3 protein is obtained, the protein may be introduced to theplant cell.

In particular embodiments, the C2c1 or C2c3 protein is mixed with guideRNA targeting the gene of interest to form a pre-assembledribonucleoprotein.

The individual components or pre-assembled ribonucleoprotein can beintroduced into the plant cell via electroporation, by bombardment withC2c1- or C2c3-associated gene product coated particles, by chemicaltransfection or by some other means of transport across a cell membrane.For instance, transfection of a plant protoplast with a pre-assembledCRISPR ribonucleoprotein has been demonstrated to ensure targetedmodification of the plant genome (as described by Woo et al. NatureBiotechnology, 2015; DOI: 10.1038/nbt.3389).

In particular embodiments, the C2c1 or C2c3 CRISPR system components areintroduced into the plant cells using nanoparticles. The components,either as protein or nucleic acid or in a combination thereof, can beuploaded onto or packaged in nanoparticles and applied to the plants(such as for instance described in WO 2008042156 and US 20130185823). Inparticular, embodiments of the invention comprise nanoparticles uploadedwith or packed with DNA molecule(s) encoding the C2c1 or C2c3 protein,DNA molecules encoding the guide RNA and/or isolated guide RNA asdescribed in WO2015089419.

Further means of introducing one or more components of the C2c1 or C2c3CRISPR system to the plant cell is by using cell penetrating peptides(CPP). Accordingly, in particular, embodiments the invention comprisescompositions comprising a cell penetrating peptide linked to the C2c1 orC2c3 protein. In particular embodiments of the present invention, theC2c1 or C2c3 protein and/or guide RNA is coupled to one or more CPPs toeffectively transport them inside plant protoplasts; see alsoRamakrishna et al., Genome Res. 2014 June; 24(6):1020-7 for Cas9 inhuman cells). In other embodiments, the C2c1 or C2c3 gene and/or guideRNA are encoded by one or more circular or non-circular DNA molecule(s)which are coupled to one or more CPPs for plant protoplast delivery. Theplant protoplasts are then regenerated to plant cells and further toplants. CPPs are generally described as short peptides of fewer than 35amino acids either derived from proteins or from chimeric sequenceswhich are capable of transporting biomolecules across cell membrane in areceptor independent manner. CPP can be cationic peptides, peptideshaving hydrophobic sequences, amphipathic peptides, peptides havingproline-rich and anti-microbial sequence, and chimeric or bipartitepeptides (Pooga and Langel 2005). CPPs are able to penetrate biologicalmembranes and as such trigger the movement of various biomoleculesacross cell membranes into the cytoplasm and to improve theirintracellular routing, and hence facilitate interaction of thebiomolecules with the target. Examples of CPP include amongst others:Tat, a nuclear transcriptional activator protein required for viralreplication by HIV type1, penetratin, Kaposi fibroblast growth factor(FGF) signal peptide sequence, integrin β3 signal peptide sequence;polyarginine peptide Args sequence, Guanine rich-molecular transporters,sweet arrow peptide, etc. . . .

Use of the C2c1 or C2c3 CRISPR System to Make Genetically ModifiedNon-Transgenic Plants

In particular embodiments, the methods described herein are used tomodify endogenous genes or to modify their expression without thepermanent introduction into the genome of the plant of any foreign gene,including those encoding CRISPR components, so as to avoid the presenceof foreign DNA in the genome of the plant. This can be of interest asthe regulatory requirements for non-transgenic plants are less rigorous.

In particular embodiments, this is ensured by transient expression ofthe C2c1 or C2c3 CRISPR components. In particular embodiments one ormore of the CRISPR components are expressed on one or more viral vectorswhich produce sufficient C2c1 or C2c3 protein and guide RNA toconsistently steadily ensure modification of a gene of interestaccording to a method described herein.

In particular embodiments, transient expression of C2c1 or C2c3 CRISPRconstructs is ensured in plant protoplasts and thus not integrated intothe genome. The limited window of expression can be sufficient to allowthe C2c1 or C2c3 CRISPR system to ensure modification of a target geneas described herein.

In particular embodiments, the different components of the C2c1 or C2c3CRISPR system are introduced in the plant cell, protoplast or planttissue either separately or in mixture, with the aid of particulatedelivering molecules such as nanoparticles or CPP molecules as describedherein above.

The expression of the C2c1 or C2c3 CRISPR components can induce targetedmodification of the genome, either by direct activity of the C2c1 orC2c3 nuclease and optionally introduction of template DNA or bymodification of genes targeted using the C2c1 or C2c3 CRISPR system asdescribed herein. The different strategies described herein above allowC2c1- or C2c3-mediated targeted genome editing without requiring theintroduction of the C2c1 or C2c3 CRISPR components into the plantgenome. Components which are transiently introduced into the plant cellare typically removed upon crossing.

Detecting Modifications in the Plant Genome-Selectable Markers

In particular embodiments, where the method involves modification of anendogenous target gene of the plant genome, any suitable method can beused to determine, after the plant, plant part or plant cell is infectedor transfected with the C2c1 or C2c3 CRISPR system, whether genetargeting or targeted mutagenesis has occurred at the target site. Wherethe method involves introduction of a transgene, a transformed plantcell, callus, tissue or plant may be identified and isolated byselecting or screening the engineered plant material for the presence ofthe transgene or for traits encoded by the transgene. Physical andbiochemical methods may be used to identify plant or plant celltransformants containing inserted gene constructs or an endogenous DNAmodification. These methods include but are not limited to: 1) Southernanalysis or PCR amplification for detecting and determining thestructure of the recombinant DNA insert or modified endogenous genes; 2)Northern blot, S1 RNase protection, primer-extension or reversetranscriptase-PCR amplification for detecting and examining RNAtranscripts of the gene constructs: 3) enzymatic assays for detectingenzyme or ribozyme activity, where such gene products are encoded by thegene construct or expression is affected by the genetic modification; 4)protein gel electrophoresis, Western blot techniques,immunoprecipitation, or enzyme-linked immunoassays, where the geneconstruct or endogenous gene products are proteins. Additionaltechniques, such as in situ hybridization, enzyme staining, andimmunostaining, also may be used to detect the presence or expression ofthe recombinant construct or detect a modification of endogenous gene inspecific plant organs and tissues. The methods for doing all theseassays are well known to those skilled in the art.

Additionally (or alternatively), the expression system encoding the C2c1or C2c3 CRISPR components is typically designed to comprise one or moreselectable or detectable markers that provide a means to isolate orefficiently select cells that contain and/or have been modified by theC2c1 or C2c3 CRISPR system at an early stage and on a large scale.

In the case of Agrobacterium-mediated transformation, the markercassette may be adjacent to or between flanking T-DNA borders andcontained within a binary vector. In another embodiment, the markercassette may be outside of the T-DNA. A selectable marker cassette mayalso be within or adjacent to the same T-DNA borders as the expressioncassette or may be somewhere else within a second T-DNA on the binaryvector (e.g., a 2 T-DNA system).

For particle bombardment or with protoplast transformation, theexpression system can comprise one or more isolated linear fragments ormay be part of a larger construct that might contain bacterialreplication elements, bacterial selectable markers or other detectableelements. The expression cassette(s) comprising the polynucleotidesencoding the guide and/or C2c1 or C2c3 may be physically linked to amarker cassette or may be mixed with a second nucleic acid moleculeencoding a marker cassette. The marker cassette is comprised ofnecessary elements to express a detectable or selectable marker thatallows for efficient selection of transformed cells.

The selection procedure for the cells based on the selectable markerwill depend on the nature of the marker gene. In particular embodiments,use is made of a selectable marker, i.e. a marker which allows a directselection of the cells based on the expression of the marker. Aselectable marker can confer positive or negative selection and isconditional or non-conditional on the presence of external substrates(Miki et al. 2004, 107(3): 193-232). Most commonly, antibiotic orherbicide resistance genes are used as a marker, whereby selection is beperformed by growing the engineered plant material on media containingan inhibitory amount of the antibiotic or herbicide to which the markergene confers resistance. Examples of such genes are genes that conferresistance to antibiotics, such as hygromycin (hpt) and kanamycin(nptII), and genes that confer resistance to herbicides, such asphosphinothricin (bar) and chlorosulfuron (als),

Transformed plants and plant cells may also be identified by screeningfor the activities of a visible marker, typically an enzyme capable ofprocessing a colored substrate (e.g., the β-glucuronidase, luciferase, Bor C1 genes). Such selection and screening methodologies are well knownto those skilled in the art.

Plant Cultures and Regeneration

In particular embodiments, plant cells which have a modified genome andthat are produced or obtained by any of the methods described herein,can be cultured to regenerate a whole plant which possesses thetransformed or modified genotype and thus the desired phenotype.Conventional regeneration techniques are well known to those skilled inthe art. Particular examples of such regeneration techniques rely onmanipulation of certain phytohormones in a tissue culture growth medium,and typically relying on a biocide and/or herbicide marker which hasbeen introduced together with the desired nucleotide sequences. Infurther particular embodiments, plant regeneration is obtained fromcultured protoplasts, plant callus, explants, organs, pollens, embryosor parts thereof (see e.g. Evans et al. (1983), Handbook of Plant CellCulture, Klee et al (1987) Ann. Rev. of Plant Phys.).

In particular embodiments, transformed or improved plants as describedherein can be self-pollinated to provide seed for homozygous improvedplants of the invention (homozygous for the DNA modification) or crossedwith non-transgenic plants or different improved plants to provide seedfor heterozygous plants. Where a recombinant DNA was introduced into theplant cell, the resulting plant of such a crossing is a plant which isheterozygous for the recombinant DNA molecule. Both such homozygous andheterozygous plants obtained by crossing from the improved plants andcomprising the genetic modification (which can be a recombinant DNA) arereferred to herein as “progeny”. Progeny plants are plants descendedfrom the original transgenic plant and containing the genomemodification or recombinant DNA molecule introduced by the methodsprovided herein. Alternatively, genetically modified plants can beobtained by one of the methods described supra using the C2c1 or C2c3enzyme whereby no foreign DNA is incorporated into the genome. Progenyof such plants, obtained by further breeding may also contain thegenetic modification. Breedings are performed by any breeding methodsthat are commonly used for different crops (e.g., Allard, Principles ofPlant Breeding, John Wiley & Sons, NY, U. of CA, Davis, Calif., 50-98(1960).

Generation of Plants with Enhanced Agronomic Traits

The C2c1 or C2c3 based CRISPR systems provided herein can be used tointroduce targeted double-strand or single-strand breaks and/or tointroduce gene activator and/or repressor systems and without beinglimitative, can be used for gene targeting, gene replacement, targetedmutagenesis, targeted deletions or insertions, targeted inversionsand/or targeted translocations. By co-expression of multiple targetingRNAs directed to achieve multiple modifications in a single cell,multiplexed genome modification can be ensured. This technology can beused to high-precision engineering of plants with improvedcharacteristics, including enhanced nutritional quality, increasedresistance to diseases and resistance to biotic and abiotic stress, andincreased production of commercially valuable plant products orheterologous compounds.

In particular embodiments, the C2c1 or C2c3 CRISPR system as describedherein is used to introduce targeted double-strand breaks (DSB) in anendogenous DNA sequence. The DSB activates cellular DNA repair pathways,which can be harnessed to achieve desired DNA sequence modificationsnear the break site. This is of interest where the inactivation ofendogenous genes can confer or contribute to a desired trait. Inparticular embodiments, homologous recombination with a templatesequence is promoted at the site of the DSB, in order to introduce agene of interest.

In particular embodiments, the C2c1 or C2c3 CRISPR system may be used asa generic nucleic acid binding protein with fusion to or being operablylinked to a functional domain for activation and/or repression ofendogenous plant genes. Exemplary functional domains may include but arenot limited to translational initiator, translational activator,translational repressor, nucleases, in particular ribonucleases, aspliceosome, beads, a light inducible/controllable domain or achemically inducible/controllable domain. Typically in theseembodiments, the C2c1 or C2c3 protein comprises at least one mutation,such that it has no more than 5% of the activity of the C2c1 or C2c3protein not having the at least one mutation; the guide RNA comprises aguide sequence capable of hybridizing to a target sequence.

The methods described herein generally result in the generation of“improved plants” in that they have one or more desirable traitscompared to the wildtype plant. In particular embodiments, the plants,plant cells or plant parts obtained are transgenic plants, comprising anexogenous DNA sequence incorporated into the genome of all or part ofthe cells of the plant. In particular embodiments, non-transgenicgenetically modified plants, plant parts or cells are obtained, in thatno exogenous DNA sequence is incorporated into the genome of any of theplant cells of the plant. In such embodiments, the improved plants arenon-transgenic. Where only the modification of an endogenous gene isensured and no foreign genes are introduced or maintained in the plantgenome, the resulting genetically modified crops contain no foreigngenes and can thus basically be considered non-transgenic. The differentapplications of the C2c1 or C2c3 CRISPR system for plant genome editingare described more in detail below:

a) Introduction of One or More Foreign Genes to Confer an AgriculturalTrait of Interest

The invention provides methods of genome editing or modifying sequencesassociated with or at a target locus of interest wherein the methodcomprises introducing a C2c1 or C2c3 effector protein complex into aplant cell, whereby the C2c1 or C2c3 effector protein complexeffectively functions to integrate a DNA insert, e.g. encoding a foreigngene of interest, into the genome of the plant cell. In preferredembodiments the integration of the DNA insert is facilitated by HR withan exogenously introduced DNA template or repair template. Typically,the exogenously introduced DNA template or repair template is deliveredtogether with the C2c1 or C2c3 effector protein complex or one componentor a polynucleotide vector for expression of a component of the complex.

The C2c1 or C2c3 CRISPR systems provided herein allow for targeted genedelivery. It has become increasingly clear that the efficiency ofexpressing a gene of interest is to a great extent determined by thelocation of integration into the genome. The present methods allow fortargeted integration of the foreign gene into a desired location in thegenome. The location can be selected based on information of previouslygenerated events or can be selected by methods disclosed elsewhereherein.

In particular embodiments, the methods provided herein include (a)introducing into the cell a C2c1 or C2c3 CRISPR complex comprising aguide RNA, comprising a direct repeat and a guide sequence, wherein theguide sequence hybridizes to a target sequence that is endogenous to theplant cell; (b) introducing into the plant cell a C2c1 or C2c3 effectormolecule which complexes with the guide RNA when the guide sequencehybridizes to the target sequence and induces a double strand break ator near the sequence to which the guide sequence is targeted; and (c)introducing into the cell a nucleotide sequence encoding an HDR repairtemplate which encodes the gene of interest and which is introduced intothe location of the DS break as a result of HDR. In particularembodiments, the step of introducing can include delivering to the plantcell one or more polynucleotides encoding C2c1 or C2c3 effector protein,the guide RNA and the repair template. In particular embodiments, thepolynucleotides are delivered into the cell by a DNA virus (e.g., ageminivirus) or an RNA virus (e.g., a tobravirus). In particularembodiments, the introducing steps include delivering to the plant cella T-DNA containing one or more polynucleotide sequences encoding theC2c1 or C2c3 effector protein, the guide RNA and the repair template,where the delivering is via Agrobacterium. The nucleic acid sequenceencoding the C2c1 or C2c3 effector protein can be operably linked to apromoter, such as a constitutive promoter (e.g., a cauliflower mosaicvirus 35S promoter), or a cell specific or inducible promoter. Inparticular embodiments, the polynucleotide is introduced bymicroprojectile bombardment. In particular embodiments, the methodfurther includes screening the plant cell after the introducing steps todetermine whether the repair template i.e. the gene of interest has beenintroduced. In particular embodiments, the methods include the step ofregenerating a plant from the plant cell. In further embodiments, themethods include cross breeding the plant to obtain a genetically desiredplant lineage. Examples of foreign genes encoding a trait of interestare listed below.

b) Editing of Endogenous Genes to Confer an Agricultural Trait ofInterest

The invention provides methods of genome editing or modifying sequencesassociated with or at a target locus of interest wherein the methodcomprises introducing a C2c1 or C2c3 effector protein complex into aplant cell, whereby the C2c1 or C2c3 complex modifies the expression ofan endogenous gene of the plant. This can be achieved in different ways,In particular embodiments, the elimination of expression of anendogenous gene is desirable and the C2c1 or C2c3 CRISPR complex is usedto target and cleave an endogenous gene so as to modify gene expression.In these embodiments, the methods provided herein include (a)introducing into the plant cell a C2c1 or C2c3 CRISPR complex comprisinga guide RNA, comprising a direct repeat and a guide sequence, whereinthe guide sequence hybridizes to a target sequence within a gene ofinterest in the genome of the plant cell; and (b) introducing into thecell a C2c1 or C2c3 effector protein, which upon binding to the guideRNA comprises a guide sequence that is hybridized to the targetsequence, ensures a double strand break at or near the sequence to whichthe guide sequence is targeted; In particular embodiments, the step ofintroducing can include delivering to the plant cell one or morepolynucleotides encoding C2c1 or C2c3 effector protein and the guideRNA.

In particular embodiments, the polynucleotides are delivered into thecell by a DNA virus (e.g., a geminivirus) or an RNA virus (e.g., atobravirus). In particular embodiments, the introducing steps includedelivering to the plant cell a T-DNA containing one or morepolynucleotide sequences encoding the C2c1 or C2c3 effector protein andthe guide RNA, where the delivering is via Agrobacterium. Thepolynucleotide sequence encoding the components of the C2c1 or C2c3CRISPR system can be operably linked to a promoter, such as aconstitutive promoter (e.g., a cauliflower mosaic virus 35S promoter),or a cell specific or inducible promoter. In particular embodiments, thepolynucleotide is introduced by microprojectile bombardment. Inparticular embodiments, the method further includes screening the plantcell after the introducing steps to determine whether the expression ofthe gene of interest has been modified. In particular embodiments, themethods include the step of regenerating a plant from the plant cell. Infurther embodiments, the methods include cross breeding the plant toobtain a genetically desired plant lineage.

In particular embodiments of the methods described above, diseaseresistant crops are obtained by targeted mutation of diseasesusceptibility genes or genes encoding negative regulators (e.g. Mlogene) of plant defense genes. In a particular embodiment,herbicide-tolerant crops are generated by targeted substitution ofspecific nucleotides in plant genes such as those encoding acetolactatesynthase (ALS) and protoporphyrinogen oxidase (PPO). In particularembodiments drought and salt tolerant crops by targeted mutation ofgenes encoding negative regulators of abiotic stress tolerance, lowamylose grains by targeted mutation of Waxy gene, rice or other grainswith reduced rancidity by targeted mutation of major lipase genes inaleurone layer, etc. In particular embodiments. A more extensive list ofendogenous genes encoding a traits of interest are listed below.

c) Modulating of Endogenous Genes by the C2c1 or C2c3 CRISPR System toConfer an Agricultural Trait of Interest

Also provided herein are methods for modulating (i.e. activating orrepressing) endogenous gene expression using the C2c1 or C2c3 proteinprovided herein. Such methods make use of distinct RNA sequence(s) whichare targeted to the plant genome by the C2c1 or C2c3 complex. Moreparticularly the distinct RNA sequence(s) bind to two or more adaptorproteins (e.g. aptamers) whereby each adaptor protein is associated withone or more functional domains and wherein at least one of the one ormore functional domains associated with the adaptor protein have one ormore activities comprising methylase activity, demethylase activity,transcription activation activity, transcription repression activity,transcription release factor activity, histone modification activity,DNA integration activity RNA cleavage activity, DNA cleavage activity ornucleic acid binding activity; The functional domains are used tomodulate expression of an endogenous plant gene so as to obtain thedesired trait. Typically, in these embodiments, the C2c1 or C2c3effector protein has one or more mutations such that it has no more than5% of the nuclease activity of the C2c1 or C2c3 effector protein nothaving the at least one mutation.

In particular embodiments, the methods provided herein include the stepsof (a) introducing into the cell a C2c1 or C2c3 CRISPR complexcomprising a guide RNA, comprising a direct repeat and a guide sequence,wherein the guide sequence hybridizes to a target sequence that isendogenous to the plant cell; (b) introducing into the plant cell a C2c1or C2c3 effector molecule which complexes with the guide RNA when theguide sequence hybridizes to the target sequence; and wherein either theguide RNA is modified to comprise a distinct RNA sequence (aptamer)binding to a functional domain and/or the C2c1 or C2c3 effector proteinis modified in that it is linked to a functional domain. In particularembodiments, the step of introducing can include delivering to the plantcell one or more polynucleotides encoding the (modified) C2c1 or C2c3effector protein and the (modified) guide RNA. The details thecomponents of the C2c1 or C2c3 CRISPR system for use in these methodsare described elsewhere herein.

In particular embodiments, the polynucleotides are delivered into thecell by a DNA virus (e.g., a geminivirus) or an RNA virus (e.g., atobravirus). In particular embodiments, the introducing steps includedelivering to the plant cell a T-DNA containing one or morepolynucleotide sequences encoding the C2c1 or C2c3 effector protein andthe guide RNA, where the delivering is via Agrobacterium. The nucleicacid sequence encoding the one or more components of the C2c1 or C2c3CRISPR system can be operably linked to a promoter, such as aconstitutive promoter (e.g., a cauliflower mosaic virus 35S promoter),or a cell specific or inducible promoter. In particular embodiments, thepolynucleotide is introduced by microprojectile bombardment. Inparticular embodiments, the method further includes screening the plantcell after the introducing steps to determine whether the expression ofthe gene of interest has been modified. In particular embodiments, themethods include the step of regenerating a plant from the plant cell. Infurther embodiments, the methods include cross breeding the plant toobtain a genetically desired plant lineage. A more extensive list ofendogenous genes encoding a traits of interest are listed below.

Use of C2c1 or C2c3 to Modify Polyploid Plants

Many plants are polyploid, which means they carry duplicate copies oftheir genomes-sometimes as many as six, as in wheat. The methodsaccording to the present invention, which make use of the C2c1 or C2c3CRISPR effector protein can be “multiplexed” to affect all copies of agene, or to target dozens of genes at once. For instance, in particularembodiments, the methods of the present invention are used tosimultaneously ensure a loss of function mutation in different genesresponsible for suppressing defences against a disease. In particularembodiments, the methods of the present invention are used tosimultaneously suppress the expression of the TaMLO-A1, TaMLO-B1 andTaMLO-D1 nucleic acid sequence in a wheat plant cell and regenerating awheat plant therefrom, in order to ensure that the wheat plant isresistant to powdery mildew (see also WO2015109752).

Exemplary Genes Conferring Agronomic Traits

As described herein above, in particular embodiments, the inventionencompasses the use of the C2c1 or C2c3 CRISPR system as describedherein for the insertion of a DNA of interest, including one or moreplant expressible gene(s). In further particular embodiments, theinvention encompasses methods and tools using the C2c1 or C2c3 system asdescribed herein for partial or complete deletion of one or more plantexpressed gene(s). In other further particular embodiments, theinvention encompasses methods and tools using the C2c1 or C2c3 system asdescribed herein to ensure modification of one or more plant-expressedgenes by mutation, substitution, insertion of one of more nucleotides.In other particular embodiments, the invention encompasses the use ofC2c1 or C2c3 CRISPR system as described herein to ensure modification ofexpression of one or more plant-expressed genes by specific modificationof one or more of the regulatory elements directing expression of saidgenes.

In particular embodiments, the invention encompasses methods whichinvolve the introduction of exogenous genes and/or the targeting ofendogenous genes and their regulatory elements, such as listed below:

1. Genes that Confer Resistance to Pests or Diseases:

-   -   Plant disease resistance genes. A plant can be transformed with        cloned resistance genes to engineer plants that are resistant to        specific pathogen strains. See, e.g., Jones et al., Science        266:789 (1994) (cloning of the tomato Cf-9 gene for resistance        to Cladosporium fulvum); Martin et al., Science 262:1432 (1993)        (tomato Pto gene for resistance to Pseudomonas syringae pv.        tomato encodes a protein kinase); Mindrinos et al., Cell        78:1089 (1994) (Arabidopsmay be RSP2 gene for resistance to        Pseudomonas syringae).    -   Genes conferring resistance to a pest, such as soybean cyst        nematode. See e.g., PCT Application WO 96/30517; PCT Application        WO 93/19181.    -   Bacillus thuringiensis proteins see, e.g., Geiser et al., Gene        48:109 (1986).    -   Lectins, see, for example, Van Damme et al., Plant Molec. Biol.        24:25 (1994.    -   Vitamin-binding protein, such as avidin, see PCT application        US93/06487, teaching the use of avidin and avidin homologues as        larvicides against insect pests.    -   Enzyme inhibitors such as protease or proteinase inhibitors or        amylase inhibitors. See, e.g., Abe et al., J. Biol. Chem.        262:16793 (1987), Huub et al., Plant Molec. Biol. 21:985        (1993)), Sumitani et al., Biosci. Biotech. Biochem.        57:1243 (1993) and U.S. Pat. No. 5,494,813.    -   Insect-specific hormones or pheromones such as ecdysteroid or        juvenile hormone, a variant thereof, a mimetic based thereon, or        an antagonist or agonist thereof. See, for example Hammock et        al., Nature 344:458 (1990).    -   Insect-specific peptides or neuropeptides which, upon        expression, disrupts the physiology of the affected pest. For        example Regan, J. Biol. Chem. 269:9 (1994) and Pratt et al.,        Biochem. Biophys. Res. Comm. 163:1243 (1989). See also U.S. Pat.        No. 5,266,317.    -   Insect-specific venom produced in nature by a snake, a wasp, or        any other organism. For example, see Pang et al., Gene 116: 165        (1992).    -   Enzymes responsible for a hyperaccumulation of a monoterpene, a        sesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid        derivative or another nonprotein molecule with insecticidal        activity.    -   Enzymes involved in the modification, including the        post-translational modification, of a biologically active        molecule; for example, a glycolytic enzyme, a proteolytic        enzyme, a lipolytic enzyme, a nuclease, a cyclase, a        transaminase, an esterase, a hydrolase, a phosphatase, a kinase,        a phosphorylase, a polymerase, an elastase, a chitinase and a        glucanase, whether natural or synthetic. See PCT application        WO93/02197, Kramer et al., Insect Biochem. Molec. Biol.        23:691 (1993) and Kawalleck et al., Plant Molec. Biol. 21:673        (1993).    -   Molecules that stimulates signal transduction. For example, see        Botella et al., Plant Molec. Biol. 24:757 (1994), and Griess et        al., Plant Physiol. 104:1467 (1994).    -   Viral-invasive proteins or a complex toxin derived therefrom.        See Beachy et al., Ann. rev. Phytopathol. 28:451 (1990).    -   Developmental-arrestive proteins produced in nature by a        pathogen or a parasite. See Lamb et al., Bio-Technology        10:1436 (1992) and Toubart et al., Plant J. 2:367 (1992).    -   A developmental-arrestive protein produced in nature by a plant.        For example, Logemann et al., Bio/Technology 10:305 (1992).    -   In plants, pathogens are often host-specific. For example, some        Fusarium species will causes tomato wilt but attacks only        tomato, and other Fusarium species attack only wheat. Plants        have existing and induced defenses to resist most pathogens.        Mutations and recombination events across plant generations lead        to genetic variability that gives rise to susceptibility,        especially as pathogens reproduce with more frequency than        plants. In plants there can be non-host resistance, e.g., the        host and pathogen are incompatible or there can be partial        resistance against all races of a pathogen, typically controlled        by many genes and/or also complete resistance to some races of a        pathogen but not to other races. Such resistance is typically        controlled by a few genes. Using methods and components of the        CRISPR-c2c1 or CRISPR-C2c3 system, a new tool now exists to        induce specific mutations in anticipation hereon. Accordingly,        one can analyze the genome of sources of resistance genes, and        in plants having desired characteristics or traits, use the        method and components of the C2c1 or C2c3 CRISPR system to        induce the rise of resistance genes. The present systems can do        so with more precision than previous mutagenic agents and hence        accelerate and improve plant breeding programs.

2. Genes Involved in Plant Diseases, Such as Those Listed in WO2013046247:

-   -   Rice diseases: Magnaporthe grisea, Cochliobolus miyabeanus,        Rhizoctonia solani, Gibberella fujikuroi; Wheat diseases:        Erysiphe graminis, Fusarium graminearum, F. avenaceum, F.        culmorum, Microdochium nivale, Puccinia striiformis, P.        graminis, P. recondita, Micronectriella nivale, Typhula sp.,        Ustilago tritici, Tilletia caries, Pseudocercosporella        herpotrichoides, Mycosphaerella graminicola, Stagonospora        nodorum, Pyrenophora tritici-repentis; Barley diseases: Erysiphe        graminis, Fusarium graminearum, F. avenaceum, F. culmorum,        Microdochium nivale, Puccinia striiformis, P. graminis, P.        hordei, Ustilago nuda, Rhynchosporium secalis, Pyrenophora        teres, Cochliobolus sativus, Pyrenophora graminea, Rhizoctonia        solani; Maize diseases: Ustilago maydis, Cochliobolus        heterostrophus, Gloeocercospora sorghi, Puccinia polysora,        Cercospora zeae-maydis, Rhizoctonia solani;    -   Citrus diseases: Diaporthe citri, Elsinoe fawcetti, Penicillium        digitatum, P. italicum, Phytophthora parasitica, Phytophthora        citrophthora; Apple diseases: Monilinia mali, Valsa        ceratosperma, Podosphaera leucotricha, Alternaria alternata        apple pathotype, Venturia inaequalis, Colletotrichum acutatum,        Phytophthora cactorum;    -   Pear diseases: Venturia nashicola, V. pirina, Alternaria        alternata Japanese pear pathotype, Gymnosporangium haraeanum,        Phytophthora cactorum;    -   Peach diseases: Monilinia fructicola, Cladosporium carpophilum,        Phomopsis sp.;    -   Grape diseases: Elsinoe ampelina, Glomerella cingulata, Uninula        necator, Phakopsora ampelopsidis, Guignardia bidwellii,        Plasmopara viticola;    -   Persimmon diseases: Gloeosporium kaki, Cercospora kaki,        Mycosphaerella nawae;    -   Gourd diseases: Colletotrichum lagenarium, Sphaerotheca        fuliginea, Mycosphaerella melonis, Fusarium oxysporum,        Pseudoperonospora cubensis, Phytophthora sp., Pythium sp.;    -   Tomato diseases: Alternaria solani, Cladosporium fulvum,        Phytophthora infestans;    -   Eggplant diseases: Phomopsis vexans, Erysiphe cichoracearum;        Brassicaceous vegetable diseases: Alternaria japonica,        Cercosporella brassicae, Plasmodiophora brassicae, Peronospora        parasitica;    -   Welsh onion diseases: Puccinia allii, Peronospora destructor;    -   Soybean diseases: Cercospora kikuchii, Elsinoe glycines,        Diaporthe phaseolorum var. sojae, Septoria glycines, Cercospora        sojina, Phakopsora pachyrhizi, Phytophthora sojae, Rhizoctonia        solani, Corynespora cassiicola, Sclerotinia sclerotiorum;    -   Kidney bean diseases: Colletotrichum lindemuthianum;    -   Peanut diseases: Cercospora personata, Cercospora arachidicola,        Sclerotium rolfsii;    -   Pea diseases pea: Erysiphe pisi;    -   Potato diseases: Alternaria solani, Phytophthora infestans,        Phytophthora erythroseptica, Spongospora subterranean, f. sp.        Subterranean:    -   Strawberry diseases: Sphaerotheca humuli, Glomerella cingulata;    -   Tea diseases: Exobasidium reticulatum, Elsinoe leucospila,        Pestalotiopsis sp., Colletotrichum theae-sinensis;    -   Tobacco diseases: Alternaria longipes, Erysiphe cichoracearum,        Colletotrichum tabacum, Peronospora tabacina, Phytophthora        nicotianae;    -   Rapeseed diseases: Sclerotinia sclerotiorum, Rhizoctonia solani;    -   Cotton diseases: Rhizoctonia solani;    -   Beet diseases: Cercospora beticola, Thanatephorus cucumeris,        Thanatephorus cucumeris, Aphanomyces cochlioides;    -   Rose diseases: Diplocarpon rosae, Sphaerotheca pannosa,        Peronospora sparsa;    -   Diseases of chrysanthemum and asteraceae: Bremia lactuca,        Septoria chrysanthemi-indici, Puccinia horiana;    -   Diseases of various plants: Pythium aphanidermatum, Pythium        debaryanum, Pythium graminicola, Pythium irregulare, Pythium        ultimum, Botrytis cinerea, Sclerotinia sclerotiorum;    -   Radish diseases: Alternaria brassicicola;    -   Zoysia diseases: Sclerotinia homeocarpa, Rhizoctonia solani;    -   Banana diseases: Mycosphaerella fijiensis, Mycosphaerella        musicola;    -   Sunflower diseases: Plasmopara halstedii;    -   Seed diseases or diseases in the initial stage of growth of        various plants caused by Aspergillus spp., Penicillium spp.,        Fusarium spp., Gibberella spp., Tricoderma spp., Thielaviopsis        spp., Rhizopus spp., Mucor spp., Corticium spp., Rhoma spp.,        Rhizoctonia spp., Diplodia spp., or the like:    -   Virus diseases of various plants mediated by Polymyxa spp.,        Olpidium spp., or the like.

3. Examples of Genes that Confer Resistance to Herbicides:

-   -   Resistance to herbicides that inhibit the growing point or        meristem, such as an imidazolinone or a sulfonylurea, for        example, by Lee et al., EMBO J. 7:1241 (1988), and Miki et al.,        Theor. Appl. Genet. 80:449 (1990), respectively.    -   Glyphosate tolerance (resistance conferred by, e.g., mutant        5-enolpyruvylshikimate-3-phosphate synthase (EPSPs) genes, aroA        genes and glyphosate acetyl transferase (GAT) genes,        respectively), or resistance to other phosphono compounds such        as by glufosinate (phosphinothricin acetyl transferase (PAT)        genes from Streptomyces species, including Streptomyces        hygroscopicus and Streptomyces viridochromogenes), and to        pyridinoxy or phenoxy propionic acids and cyclohexones by ACCase        inhibitor-encoding genes. See, for example, U.S. Pat. Nos.        4,940,835 and 6,248,876, 4,769,061, EP No. 0 333 033 and U.S.        Pat. No. 4,975,374. See also EP No. 0242246, DeGreef et al.,        Bio/Technology 7:61 (1989), Marshall et al., Theor. Appl. Genet.        83:435 (1992), WO 2005012515 to Castle et. al. and WO        2005107437.    -   Resistance to herbicides that inhibit photosynthesis, such as a        triazine (psbA and gs+ genes) or a benzonitrile (nitrilase        gene), and glutathione S-transferase in Przibila et al., Plant        Cell 3:169 (1991), U.S. Pat. No. 4,810,648, and Hayes et al.,        Biochem. J. 285: 173 (1992).    -   Genes encoding Enzymes detoxifying the herbicide or a mutant        glutamine synthase enzyme that is resistant to inhibition, e.g.        n U.S. patent application Ser. No. 11/760,602. Or a detoxifying        enzyme is an enzyme encoding a phosphinothricin        acetyltransferase (such as the bar or pat protein from        Streptomyces species). Phosphinothricin acetyltransferases are        for example described in U.S. Pat. Nos. 5,561,236; 5,648,477;        5,646,024; 5,273,894; 5,637,489; 5,276,268; 5,739,082; 5,908,810        and 7,112,665.    -   Hydroxyphenylpyruvatedioxygenases (HPPD) inhibitors, ie        naturally occurring HPPD resistant enzymes, or genes encoding a        mutated or chimeric HPPD enzyme as described in WO 96/38567, WO        99/24585, and WO 99/24586, WO 2009/144079, WO 2002/046387, or        U.S. Pat. No. 6,768,044.

4. Examples of Genes Involved in Abiotic Stress Tolerance:

-   -   Transgene capable of reducing the expression and/or the activity        of poly(ADP-ribose) polymerase (PARP) gene in the plant cells or        plants as described in WO 00/04173 or, WO 2006/045633.    -   Transgenes capable of reducing the expression and/or the        activity of the PARG encoding genes of the plants or plants        cells, as described e.g. in WO 2004/090140.    -   Transgenes coding for a plant-functional enzyme of the        nicotineamide adenine dinucleotide salvage synthesis pathway        including nicotinamidase, nicotinate phosphoribosyltransferase,        nicotinic acid mononucleotide adenyl transferase, nicotinamide        adenine dinucleotide synthetase or nicotine amide        phosphorybosyltransferase as described e.g. in EP 04077624.7, WO        2006/133827, PCT/EP07/002,433, EP 1999263, or WO 2007/107326.    -   Enzymes involved in carbohydrate biosynthesis include those        described in e.g. EP 0571427, WO 95/04826, EP 0719338, WO        96/15248, WO 96/19581, WO 96/27674, WO 97/11188, WO 97/26362, WO        97/32985, WO 97/42328, WO 97/44472, WO 97/45545, WO 98/27212, WO        98/40503, WO 99/58688, WO 99/58690, WO 99/58654, WO 00/08184, WO        00/08185, WO 00/08175, WO 00/28052, WO 00/77229, WO 01/12782, WO        01/12826, WO 02/101059, WO 03/071860, WO 2004/056999, WO        2005/030942, WO 2005/030941, WO 2005/095632, WO 2005/095617, WO        2005/095619, WO 2005/095618, WO 2005/123927, WO 2006/018319, WO        2006/103107, WO 2006/108702, WO 2007/009823, WO 00/22140, WO        2006/063862, WO 2006/072603, WO 02/034923, EP 06090134.5, EP        06090228.5, EP 06090227.7, EP 07090007.1, EP 07090009.7, WO        01/14569, WO 02/79410, WO 03/33540, WO 2004/078983, WO 01/19975,        WO 95/26407, WO 96/34968, WO 98/20145, WO 99/12950, WO 99/66050,        WO 99/53072, U.S. Pat. No. 6,734,341, WO 00/11192, WO 98/22604,        WO 98/32326, WO 01/98509, WO 01/98509, WO 2005/002359, U.S. Pat.        Nos. 5,824,790, 6,013,861, WO 94/04693, WO 94/09144, WO        94/11520, WO 95/35026 or WO 97/20936 or enzymes involved in the        production of polyfructose, especially of the inulin and        levan-type, as disclosed in EP 0663956, WO 96/01904, WO        96/21023, WO 98/39460, and WO 99/24593, the production of        alpha-1,4-glucans as disclosed in WO 95/31553, US 2002031826,        U.S. Pat. Nos. 6,284,479, 5,712,107, WO 97/47806, WO 97/47807,        WO 97/47808 and WO 00/14249, the production of alpha-1,6        branched alpha-1,4-glucans, as disclosed in WO 00/73422, the        production of alternan, as disclosed in e.g. WO 00/47727, WO        00/73422, EP 06077301.7, U.S. Pat. No. 5,908,975 and EP 0728213,        the production of hyaluronan, as for example disclosed in WO        2006/032538, WO 2007/039314, WO 2007/039315, WO 2007/039316, JP        2006304779, and WO 2005/012529.    -   Genes that improve drought resistance. For example, WO        2013122472 discloses that the absence or reduced level of        functional Ubiquitin Protein Ligase protein (UPL) protein, more        specifically, UPL3, leads to a decreased need for water or        improved resistance to drought of said plant. Other examples of        transgenic plants with increased drought tolerance are disclosed        in, for example, US 2009/0144850, US 2007/0266453, and WO        2002/083911. US2009/0144850 describes a plant displaying a        drought tolerance phenotype due to altered expression of a DR02        nucleic acid. US 2007/0266453 describes a plant displaying a        drought tolerance phenotype due to altered expression of a DR03        nucleic acid and WO 2002/08391 1 describes a plant having an        increased tolerance to drought stress due to a reduced activity        of an ABC transporter which is expressed in guard cells. Another        example is the work by Kasuga and co-authors (1999), who        describe that overexpression of cDNA encoding DREB1 A in        transgenic plants activated the expression of many stress        tolerance genes under normal growing conditions and resulted in        improved tolerance to drought, salt loading, and freezing.        However, the expression of DREB1A also resulted in severe growth        retardation under normal growing conditions (Kasuga (1999) Nat        Biotechnol 17(3) 287-291).

In further particular embodiments, crop plants can be improved byinfluencing specific plant traits. For example, by developingpesticide-resistant plants, improving disease resistance in plants,improving plant insect and nematode resistance, improving plantresistance against parasitic weeds, improving plant drought tolerance,improving plant nutritional value, improving plant stress tolerance,avoiding self-pollination, plant forage digestibility biomass, grainyield etc. A few specific non-limiting examples are providedhereinbelow.

In addition to targeted mutation of single genes, C2c1 or C2c3 CRISPRcomplexes can be designed to allow targeted mutation of multiple genes,deletion of chromosomal fragment, site-specific integration oftransgene, site-directed mutagenesis in vivo, and precise genereplacement or allele swapping in plants. Therefore, the methodsdescribed herein have broad applications in gene discovery andvalidation, mutational and cisgenic breeding, and hybrid breeding. Theseapplications facilitate the production of a new generation ofgenetically modified crops with various improved agronomic traits suchas herbicide resistance, disease resistance, abiotic stress tolerance,high yield, and superior quality.

Use of C2c1 or C2e3 Gene to Create Male Sterile Plants

Hybrid plants typically have advantageous agronomic traits compared toinbred plants. However, for self-pollinating plants, the generation ofhybrids can be challenging. In different plant types, genes have beenidentified which are important for plant fertility, more particularlymale fertility. For instance, in maize, at least two genes have beenidentified which are important in fertility (Amitabh MohantyInternational Conference on New Plant Breeding Molecular TechnologiesTechnology Development And Regulation, Oct. 9-10, 2014, Jaipur, India;Svitashev et al. Plant Physiol. 2015 October; 169(2):931-45; Djukanovicet al. Plant J. 2013 December; 76(5):888-99). The methods providedherein can be used to target genes required for male fertility so as togenerate male sterile plants which can easily be crossed to generatehybrids. In particular embodiments, the C2c1 or C2c3 CRISPR systemprovided herein is used for targeted mutagenesis of the cytochromeP450-like gene (MS26) or the meganuclease gene (MS45) thereby conferringmale sterility to the maize plant. Maize plants which are as suchgenetically altered can be used in hybrid breeding programs.

Increasing the Fertility Stage in Plants

In particular embodiments, the methods provided herein are used toprolong the fertility stage of a plant such as of a rice plant. Forinstance, a rice fertility stage gene such as Ehd3 can be targeted inorder to generate a mutation in the gene and plantlets can be selectedfor a prolonged regeneration plant fertility stage (as described in CN104004782).

Use of C2c1 or C2e3 to Generate Genetic Variation in a Crop of Interest

The availability of wild germplasm and genetic variations in crop plantsis the key to crop improvement programs, but the available diversity ingermplasms from crop plants is limited. The present invention envisagesmethods for generating a diversity of genetic variations in a germplasmof interest. In this application of the C2c1 or C2c3 CRISPR system alibrary of guide RNAs targeting different locations in the plant genomeis provided and is introduced into plant cells together with the C2c1 orC2c3 effector protein. In this way a collection of genome-scale pointmutations and gene knock-outs can be generated. In particularembodiments, the methods comprise generating a plant part or plant fromthe cells so obtained and screening the cells for a trait of interest.The target genes can include both coding and non-coding regions. Inparticular embodiments, the trait is stress tolerance and the method isa method for the generation of stress-tolerant crop varieties.

Use of C2c1 or C2e3 to Affect Fruit-Ripening

Ripening is a normal phase in the maturation process of fruits andvegetables. Only a few days after it starts it renders a fruit orvegetable inedible. This process brings significant losses to bothfarmers and consumers. In particular embodiments, the methods of thepresent invention are used to reduce ethylene production. This isensured by ensuring one or more of the following: a. Suppression of ACCsynthase gene expression. ACC (1-aminocyclopropane-1-carboxylic acid)synthase is the enzyme responsible for the conversion ofS-adenosylmethionine (SAM) to ACC; the second to the last step inethylene biosynthesis. Enzyme expression is hindered when an antisense(“mirror-image”) or truncated copy of the synthase gene is inserted intothe plant's genome; b. Insertion of the ACC deaminase gene. The genecoding for the enzyme is obtained from Pseudomonas chlororaphis, acommon nonpathogenic soil bacterium. It converts ACC to a differentcompound thereby reducing the amount of ACC available for ethyleneproduction; c. Insertion of the SAM hydrolase gene. This approach issimilar to ACC deaminase wherein ethylene production is hindered whenthe amount of its precursor metabolite is reduced; in this case SAM isconverted to homoserine. The gene coding for the enzyme is obtained fromE. coli T3 bacteriophage and d. Suppression of ACC oxidase geneexpression. ACC oxidase is the enzyme which catalyzes the oxidation ofACC to ethylene, the last step in the ethylene biosynthetic pathway.Using the methods described herein, down regulation of the ACC oxidasegene results in the suppression of ethylene production, thereby delayingfruit ripening. In particular embodiments, additionally or alternativelyto the modifications described above, the methods described herein areused to modify ethylene receptors, so as to interfere with ethylenesignals obtained by the fruit. In particular embodiments, expression ofthe ETR1 gene, encoding an ethylene binding protein is modified, moreparticularly suppressed. In particular embodiments, additionally oralternatively to the modifications described above, the methodsdescribed herein are used to modify expression of the gene encodingPolygalacturonase (PG), which is the enzyme responsible for thebreakdown of pectin, the substance that maintains the integrity of plantcell walls. Pectin breakdown occurs at the start of the ripening processresulting in the softening of the fruit. Accordingly, in particularembodiments, the methods described herein are used to introduce amutation in the PG gene or to suppress activation of the PG gene inorder to reduce the amount of PG enzyme produced thereby delaying pectindegradation.

Thus in particular embodiments, the methods comprise the use of the C2c1or C2c3 CRISPR system to ensure one or more modifications of the genomeof a plant cell such as described above, and regenerating a planttherefrom. In particular embodiments, the plant is a tomato plant.

Increasing Storage Life of Plants

In particular embodiments, the methods of the present invention are usedto modify genes involved in the production of compounds which affectstorage life of the plant or plant part. More particularly, themodification is in a gene that prevents the accumulation of reducingsugars in potato tubers. Upon high-temperature processing, thesereducing sugars react with free amino acids, resulting in brown,bitter-tasting products and elevated levels of acrylamide, which is apotential carcinogen. In particular embodiments, the methods providedherein are used to reduce or inhibit expression of the vacuolarinvertase gene (VInv), which encodes a protein that breaks down sucroseto glucose and fructose (Clasen et al. DOI: 10.1111/pbi.12370).

The Use of the C2c1 or C293 CRISPR System to Ensure a Value Added Trait

In particular embodiments the C2c1 or C2c3 CRISPR system is used toproduce nutritionally improved agricultural crops. In particularembodiments, the methods provided herein are adapted to generate“functional foods”, i.e. a modified food or food ingredient that mayprovide a health benefit beyond the traditional nutrients it containsand/or “nutraceutical”, i.e. substances that may be considered a food orpart of a food and provides health benefits, including the preventionand treatment of disease. In particular embodiments, the nutraceuticalis useful in the prevention and/or treatment of one or more of cancer,diabetes, cardiovascular disease, and hypertension.

Examples of nutritionally improved crops include (Newell-McGloughlin,Plant Physiology, July 2008, Vol. 147, pp. 939-953):

-   -   modified protein quality, content and/or amino acid composition,        such as have been described for Bahiagrass (Luciani et al. 2005,        Florida Genetics Conference Poster), Canola (Roesler et al.,        1997, Plant Physiol 113 75-81), Maize (Cromwell et al, 1967,        1969 J Anim Sci 26 1325-1331, O'Quin et al. 2000 J Anim Sci 78        2144-2149, Yang et al. 2002, Transgenic Res 11 11-20, Young et        al. 2004, Plant J 38 910-922), Potato (Yu J and Ao, 1997 Acta        Bot Sin 39 329-334; Chakraborty et al. 2000, Proc Natl Acad Sci        USA 97 3724-3729; Li et al. 2001) Chin Sci Bull 46 482-484, Rice        (Katsube et al. 1999, Plant Physiol 120 1063-1074), Soybean        (Dinkins et al. 2001, Rapp 2002, In Vitro Cell Dev Biol Plant 37        742-747), Sweet Potato (Egnin and Prakash 1997, In Vitro Cell        Dev Biol 33 52A).    -   essential amino acid content, such as has been described for        Canola (Falco et al. 1995, Bio/Technology 13 577-582), Lupin        (White et al. 2001, J Sci Food Agric 81 147-154), Maize (Lai and        Messing, 2002, Agbios 2008 GM crop database (Mar. 11, 2008)),        Potato (Zeh et al. 2001, Plant Physiol 127 792-802), Sorghum        (Zhao et al. 2003, Kluwer Academic Publishers, Dordrecht, The        Netherlands, pp 413-416), Soybean (Falco et al. 1995        Bio/Technology 13 577-582; Galili et al. 2002 Crit Rev Plant Sci        21 167-204).    -   Oils and Fatty acids such as for Canola (Dehesh et al. (1996)        Plant J 9 167-172 [PubMed]; Del Vecchio (1996) INFORM        International News on Fats, Oils and Related Materials 7        230-243; Roesler et al. (1997) Plant Physiol 113 75-81 [PMC free        article][PubMed]; Froman and Ursin (2002, 2003) Abstracts of        Papers of the American Chemical Society 223 U35; James et        al. (2003) Am J Clin Nutr 77 1140-1145 [PubMed]; Agbios (2008,        above); coton (Chapman et al. (2001). J Am Oil Chem Soc 78        941-947; Liu et al. (2002) J Am Coll Nutr 21 205S-211S [PubMed];        O'Neill (2007) Australian Life Scientist.        www.biotechnews.com.au/ndex.php/id;866694817;fp;4;fpid;2 (Jun.        17, 2008), Linseed (Abbadi et al., 2004, Plant Cell 16:        2734-2748), Maize (Young et al., 2004, Plant J 38 910-922), oil        palm (Jalani et al. 1997, J Am Oil Chem Soc 74 1451-1455;        Parveez, 2003, AgBiotechNet 113 1-8), Rice (Anai et al., 2003,        Plant Cell Rep 21 988-992), Soybean (Reddy and Thomas, 1996, Nat        Biotechnol 14 639-642; Kinney and Kwolton, 1998, Blackie        Academic and Professional, London, pp 193-213), Sunflower        (Arcadia, Biosciences 2008)    -   Carbohydrates, such as Fructans described for Chicory        (Smeekens (1997) Trends Plant Sci 2 286-287, Sprenger et        al. (1997) FEBS Lett 400 355-358, Sevenier et al. (1998) Nat        Biotechnol 16 843-846), Maize (Caimi et al. (1996) Plant Physiol        110 355-363), Potato (Hellwege et al., 1997 Plant J 12        1057-1065), Sugar Beet (Smeekens et al. 1997, above), Inulin,        such as described for Potato (Hellewege et al. 2000, Proc Natl        Acad Sci USA 97 8699-8704), Starch, such as described for Rice        (Schwall et al. (2000) Nat Biotechnol 18 551-554, Chiang et        al. (2005) Mol Breed 15 125-143),    -   Vitamins and carotenoids, such as described for Canola (Shintani        and DellaPenna (1998) Science 282 2098-2100), Maize (Rocheford        et al. (2002). J Am Coll Nutr 21 191S-198S, Cahoon et al. (2003)        Nat Biotechnol 21 1082-1087, Chen et al. (2003) Proc Natl Acad        Sci USA 100 3525-3530), Mustardseed (Shewmaker et al. (1999)        Plant J 20 401-412, Potato (Ducreux et al., 2005, J Exp Bot 56        81-89), Rice (Ye et al. (2000) Science 287 303-305, Strawberry        (Agius et al. (2003), Nat Biotechnol 21 177-181), Tomato (Rosati        et al. (2000) Plant J 24 413-419, Fraser et al. (2001) J Sci        Food Agric 81 822-827, Mehta et al. (2002) Nat Biotechnol 20        613-618, Diaz de la Garza et al. (2004) Proc Natl Acad Sci USA        101 13720-13725, Enfissi et al. (2005) Plant Biotechnol J 3        17-27, DellaPenna (2007) Proc Natl Acad Sci USA 104 3675-3676.    -   Functional secondary metabolites, such as described for Apple        (stilbenes, Szankowski et al. (2003) Plant Cell Rep 22:        141-149), Alfalfa (resveratrol, Hipskind and Paiva (2000) Mol        Plant Microbe Interact 13 551-562), Kiwi (resveratrol, Kobayashi        et al. (2000) Plant Cell Rep 19 904-910), Maize and Soybean        (flavonoids, Yu et al. (2000) Plant Physiol 124 781-794), Potato        (anthocyanin and alkaloid glycoside, Lukaszewicz et al. (2004) J        Agric Food Chem 52 1526-1533), Rice (flavonoids & resveratrol,        Stark-Lorenzen et al. (1997) Plant Cell Rep 16 668-673, Shin et        al. (2006) Plant Biotechnol J 4 303-315), Tomato (+resveratrol,        chlorogenic acid, flavonoids, stilbene; Rosati et al. (2000)        above, Muir et al. (2001) Nature 19 470-474, Niggeweg et        al. (2004) Nat Biotechnol 22 746-754, Giovinazzo et al. (2005)        Plant Biotechnol J 3 57-69), wheat (caffeic and ferulic acids,        resveratrol; United Press International (2002)); and    -   Mineral availabilities such as described for Alfalfa (phytase,        Austin-Phillips et al. (1999)        www.molecularfarming.com/nonmedical.html), Lettuse (iron, Goto        et al. (2000) Theor Appl Genet 100 658-664), Rice (iron, Lucca        et al. (2002) J Am Coll Nutr 21 184S-190S), Maize, Soybean and        wheate (phytase, Drakakaki et al. (2005) Plant Mol Biol 59        869-880, Denbow et al. (1998) Poult Sci 77 878-881,        Brinch-Pedersen et al. (2000) Mol Breed 6 195-206).

In particular embodiments, the value-added trait is related to theenvisaged health benefits of the compounds present in the plant. Forinstance, in particular embodiments, the value-added crop is obtained byapplying the methods of the invention to ensure the modification of orinduce/increase the synthesis of one or more of the following compounds:

-   -   Carotenoids, such as α-Carotene present in carrots which        Neutralizes free radicals that may cause damage to cells or        β-Carotene present in various fruits and vegetables which        neutralizes free radicals    -   Lutein present in green vegetables which contributes to        maintenance of healthy vision    -   Lycopene present in tomato and tomato products, which is        believed to reduce the risk of prostate cancer    -   Zeaxanthin, present in citrus and maize, which contributes to        maintenance of healthy vision    -   Dietary fiber such as insoluble fiber present in wheat bran        which may reduce the risk of breast and/or colon cancer and        β-Glucan present in oat, soluble fiber present in Psyllium and        whole cereal grains which may reduce the risk of cardiovascular        disease (CVD)    -   Fatty acids, such as ω-3 fatty acids which may reduce the risk        of CVD and improve mental and visual functions, Conjugated        linoleic acid, which may improve body composition, may decrease        risk of certain cancers and GLA which may reduce inflammation        risk of cancer and CVD, may improve body composition    -   Flavonoids such as Hydroxycinnamates, present in wheat which        have Antioxidant-like activities, may reduce risk of        degenerative diseases, flavonols, catechins and tannins present        in fruits and vegetables which neutralize free radicals and may        reduce risk of cancer    -   Glucosinolates, indoles, isothiocyanates, such as Sulforaphane,        present in Cruciferous vegetables (broccoli, kale), horseradish,        which neutralize free radicals, may reduce risk of cancer    -   Phenolics, such as stilbenes present in grape which May reduce        risk of degenerative diseases, heart disease, and cancer, may        have longevity effect and caffeic acid and ferulic acid present        in vegetables and citrus which have Antioxidant-like activities,        may reduce risk of degenerative diseases, heart disease, and eye        disease, and epicatechin present in cacao which has        Antioxidant-like activities, may reduce risk of degenerative        diseases and heart disease    -   Plant stanols/sterols present in maize, soy, wheat and wooden        oils which May reduce risk of coronary heart disease by lowering        blood cholesterol levels    -   Fructans, inulins, fructo-oligosaccharides present in Jerusalem        artichoke, shallot, onion powder which may improve        gastrointestinal health    -   Saponins present in soybean, which may lower LDL cholesterol    -   Soybean protein present in soybean which may reduce risk of        heart disease    -   Phytoestrogens such as isoflavones present in soybean which May        reduce menopause symptoms, such as hot flashes, may reduce        osteoporosis and CVD and lignans present in flax, rye and        vegetables, which May protect against heart disease and some        cancers, may lower LDL cholesterol, total cholesterol.    -   Sulfides and thiols such as diallyl sulphide present in onion,        garlic, olive, leek and scallon and Allyl methyl trisulfide,        dithiolthiones present in cruciferous vegetables which may lower        LDL cholesterol, helps to maintain healthy immune system    -   Tannins, such as proanthocyanidins, present in cranberry, cocoa,        which may improve urinary tract health, may reduce risk of CVD        and high blood pressure    -   Etc.

In addition, the methods of the present invention also envisagemodifying protein/starch functionality, shelf life, taste/aesthetics,fiber quality, and allergen, antinutrient, and toxin reduction traits.

Accordingly, the invention encompasses methods for producing plants withnutritional added value, said methods comprising introducing into aplant cell a gene encoding an enzyme involved in the production of acomponent of added nutritional value using the C2c1 or C2c3 CRISPRsystem as described herein and regenerating a plant from said plantcell, said plant characterized in an increase expression of saidcomponent of added nutritional value. In particular embodiments, theC2c1 or C2c3 CRISPR system is used to modify the endogenous synthesis ofthese compounds indirectly, e.g. by modifying one or more transcriptionfactors that controls the metabolism of this compound. Methods forintroducing a gene of interest into a plant cell and/or modifying anendogenous gene using the C2c1 or C2c3 CRISPR system are describedherein above.

Some specific examples of modifications in plants that have beenmodified to confer value-added traits are: plants with modified fattyacid metabolism, for example, by transforming a plant with an antisensegene of stearyl-ACP desaturase to increase stearic acid content of theplant. See Knultzon et al., Proc. Natl. Acad. Sci. U.S.A. 89:2624(1992). Another example involves decreasing phytate content, for exampleby cloning and then reintroducing DNA associated with the single allelewhich may be responsible for maize mutants characterized by low levelsof phytic acid. See Raboy et al, Maydica 35:383 (1990).

Similarly, expression of the maize (Zea mays) Tfs C1 and R, whichregulate the production of flavonoids in maize aleurone layers under thecontrol of a strong promoter, resulted in a high accumulation rate ofanthocyanins in Arabidopsis (Arabidopsis thaliana), presumably byactivating the entire pathway (Bruce et al., 2000, Plant Cell 12:65-80).DellaPenna (Welsch et al., 2007 Annu Rev Plant Biol 57: 711-738) foundthat Tf RAP2.2 and its interacting partner SINAT2 increasedcarotenogenesis in Arabidopsis leaves. Expressing the Tf Dof1 inducedthe up-regulation of genes encoding enzymes for carbon skeletonproduction, a marked increase of amino acid content, and a reduction ofthe Glc level in transgenic Arabidopsis (Yanagisawa, 2004 Plant CellPhysiol 45: 386-391), and the DOF Tf AtDof1.1 (OBP2) up-regulated allsteps in the glucosinolate biosynthetic pathway in Arabidopsis (Skiryczet al., 2006 Plant J 47: 10-24).

Reducing Allergen in Plants

In particular embodiments the methods provided herein are used togenerate plants with a reduced level of allergens, making them safer forthe consumer. In particular embodiments, the methods comprise modifyingexpression of one or more genes responsible for the production of plantallergens. For instance, in particular embodiments, the methods comprisedown-regulating expression of a Lol p5 gene in a plant cell, such as aryegrass plant cell and regenerating a plant therefrom so as to reduceallergenicity of the pollen of said plant (Bhalla et al. 1999, Proc.Natl. Acad. Sci. USA Vol. 96: 11676-11680).

Peanut allergies and allergies to legumes generally are a real andserious health concern. The C2c1 or C2c3 effector protein system of thepresent invention can be used to identify and then edit or silence genesencoding allergenic proteins of such legumes. Without limitation as tosuch genes and proteins, Nicolaou et al. identifies allergenic proteinsin peanuts, soybeans, lentils, peas, lupin, green beans, and mung beans.See, Nicolaou et al., Current Opinion in Allergy and Clinical Immunology2011; 11(3):222).

Screening Methods for Endogenous Genes of Interest

The methods provided herein further allow the identification of genes ofvalue encoding enzymes involved in the production of a component ofadded nutritional value or generally genes affecting agronomic traits ofinterest, across species, phyla, and plant kingdom. By selectivelytargeting e.g. genes encoding enzymes of metabolic pathways in plantsusing the C2c1 or C2c3 CRISPR system as described herein, the genesresponsible for certain nutritional aspects of a plant can beidentified. Similarly, by selectively targeting genes which may affect adesirable agronomic trait, the relevant genes can be identified.Accordingly, the present invention encompasses screening methods forgenes encoding enzymes involved in the production of compounds with aparticular nutritional value and/or agronomic traits.

Further Applications of the C2c1 or C293 CRISPR System in Plants andYeasts

Use of C2c1 or C2c3 CRISPR System in Biofuel Production

The term “biofuel” as used herein is an alternative fuel made from plantand plant-derived resources. Renewable biofuels can be extracted fromorganic matter whose energy has been obtained through a process ofcarbon fixation or are made through the use or conversion of biomass.This biomass can be used directly for biofuels or can be converted toconvenient energy containing substances by thermal conversion, chemicalconversion, and biochemical conversion. This biomass conversion canresult in fuel in solid, liquid, or gas form. There are two types ofbiofuels: bioethanol and biodiesel. Bioethanol is mainly produced by thesugar fermentation process of cellulose (starch), which is mostlyderived from maize and sugar cane. Biodiesel on the other hand is mainlyproduced from oil crops such as rapeseed, palm, and soybean. Biofuelsare used mainly for transportation.

Enhancing Plant Properties for Biofuel Production

In particular embodiments, the methods using the C2c1 or C2c3 CRISPRsystem as described herein are used to alter the properties of the cellwall in order to facilitate access by key hydrolysing agents for a moreefficient release of sugars for fermentation. In particular embodiments,the biosynthesis of cellulose and/or lignin are modified. Cellulose isthe major component of the cell wall. The biosynthesis of cellulose andlignin are co-regulated. By reducing the proportion of lignin in a plantthe proportion of cellulose can be increased. In particular embodiments,the methods described herein are used to downregulate ligninbiosynthesis in the plant so as to increase fermentable carbohydrates.More particularly, the methods described herein are used to downregulateat least a first lignin biosynthesis gene selected from the groupconsisting of 4-coumarate 3-hydroxylase (C3H), phenylalanineammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), hydroxycinnamoyltransferase (HCT), caffeic acid O-methyltransferase (COMT), caffeoyl CoA3-O-methyltransferase (CCoAOMT), ferulate 5-hydroxylase (F5H), cinnamylalcohol dehydrogenase (CAD), cinnamoyl CoA-reductase (CCR),4-coumarate-CoA ligase (4CL), monolignol-lignin-specificglycosyltransferase, and aldehyde dehydrogenase (ALDH) as disclosed inWO 2008064289 A2.

In particular embodiments, the methods described herein are used toproduce plant mass that produces lower levels of acetic acid duringfermentation (see also WO 2010096488). More particularly, the methodsdisclosed herein are used to generate mutations in homologs to CasiL toreduce polysaccharide acetylation.

Modifying Yeast for Biofuel Production

In particular embodiments, the C2c1 or C2c3 enzyme provided herein isused for bioethanol production by recombinant micro-organisms. Forinstance, C2c1 or C2c3 can be used to engineer micro-organisms, such asyeast, to generate biofuel or biopolymers from fermentable sugars andoptionally to be able to degrade plant-derived lignocellulose derivedfrom agricultural waste as a source of fermentable sugars. Moreparticularly, the invention provides methods whereby the C2c1 or C2c3CRISPR complex is used to introduce foreign genes required for biofuelproduction into micro-organisms and/or to modify endogenous genes whymay interfere with the biofuel synthesis. More particularly the methodsinvolve introducing into a micro-organism such as a yeast one or morenucleotide sequence encoding enzymes involved in the conversion ofpyruvate to ethanol or another product of interest. In particularembodiments the methods ensure the introduction of one or more enzymeswhich allows the micro-organism to degrade cellulose, such as acellulase. In yet further embodiments, the C2c1 or C2c3 CRISPR complexis used to modify endogenous metabolic pathways which compete with thebiofuel production pathway.

Accordingly, in more particular embodiments, the methods describedherein are used to modify a micro-organism as follows:

-   -   to introduce at least one heterologous nucleic acid or increase        expression of at least one endogenous nucleic acid encoding a        plant cell wall degrading enzyme, such that said micro-organism        is capable of expressing said nucleic acid and of producing and        secreting said plant cell wall degrading enzyme; Ser. No.    -   to introduce at least one heterologous nucleic acid or increase        expression of at least one endogenous nucleic acid encoding an        enzyme that converts pyruvate to acetaldehyde optionally        combined with at least one heterologous nucleic acid encoding an        enzyme that converts acetaldehyde to ethanol such that said host        cell is capable of expressing said nucleic acid; and/or    -   to modify at least one nucleic acid encoding for an enzyme in a        metabolic pathway in said host cell, wherein said pathway        produces a metabolite other than acetaldehyde from pyruvate or        ethanol from acetaldehyde, and wherein said modification results        in a reduced production of said metabolite, or to introduce at        least one nucleic acid encoding for an inhibitor of said enzyme.        Modifying Algae and Plants for Production of Vegetable Oils or        Biofuels

Transgenic algae or other plants such as rape may be particularly usefulin the production of vegetable oils or biofuels such as alcohols(especially methanol and ethanol), for instance. These may be engineeredto express or overexpress high levels of oil or alcohols for use in theoil or biofuel industries.

According to particular embodiments of the invention, the C2c1 or C2c3CRISPR system is used to generate lipid-rich diatoms which are useful inbiofuel production.

In particular embodiments it is envisaged to specifically modify genesthat are involved in the modification of the quantity of lipids and/orthe quality of the lipids produced by the algal cell. Examples of genesencoding enzymes involved in the pathways of fatty acid synthesis canencode proteins having for instance acetyl-CoA carboxylase, fatty acidsynthase, 3-ketoacyl_acyl-carrier protein synthase III,glycerol-3-phosphate dehydrogenase (G3PDH), Enoyl-acyl carrier proteinreductase (Enoyl-ACP-reductase), glycerol-3-phosphate acyltransferase,lysophosphatidic acyl transferase or diacylglycerol acyltransferase,phospholipid:diacylglycerol acyltransferase, phosphatidate phosphatase,fatty acid thioesterase such as palmitoyl protein thioesterase, or malicenzyme activities. In further embodiments it is envisaged to generatediatoms that have increased lipid accumulation. This can be achieved bytargeting genes that decrease lipid catabolisation. Of particularinterest for use in the methods of the present invention are genesinvolved in the activation of both triacylglycerol and free fatty acids,as well as genes directly involved in β-oxidation of fatty acids, suchas acyl-CoA synthetase, 3-ketoacyl-CoA thiolase, acyl-CoA oxidaseactivity and phosphoglucomutase. The C2c1 or C2c3 CRISPR system andmethods described herein can be used to specifically activate such genesin diatoms as to increase their lipid content.

Organisms such as microalgae are widely used for synthetic biology.Stovicek et al. (Metab. Eng. Comm., 2015; 2:13 describes genome editingof industrial yeast, for example, Saccharomyces cerevisiae, toefficiently produce robust strains for industrial production. Stovicekused a CRISPR-Cas9 system codon-optimized for yeast to simultaneouslydisrupt both alleles of an endogenous gene and knock in a heterologousgene. Cas9 and gRNA were expressed from genomic or episomal 2μ-basedvector locations. The authors also showed that gene disruptionefficiency could be improved by optimization of the levels of Cas9 andgRNA expression. Hlavová et al. (Biotechnol. Adv. 2015) discussesdevelopment of species or strains of microalgae using techniques such asCRISPR to target nuclear and chloroplast genes for insertionalmutagenesis and screening. The methods of Stovicek and Hlavová may beapplied to the C2c1 or C2c3 effector protein system of the presentinvention.

U.S. Pat. No. 8,945,839 describes a method for engineering Micro-Algae(Chlamydomonas reinhardtii cells) species) using Cas9. Using similartools, the methods of the C2c1 or C2c3 CRISPR system described hereincan be applied on Chlamydomonas species and other algae. In particularembodiments, C2c1 or C2c3 and guide RNA are introduced in algaeexpressed using a vector that expresses C2c1 or C2c3 under the controlof a constitutive promoter such as Hsp70A-Rbc S2 or Beta2-tubulin. GuideRNA will be delivered using a vector containing T7 promoter.Alternatively, C2c1 or C2c3 mRNA and in vitro transcribed guide RNA canbe delivered to algal cells. Electroporation protocol follows standardrecommended protocol from the GeneArt Chlamydomonas Engineering kit.

The Use of C2c1 or C2c3 in the Generation of Micro-Organisms Capable ofFatty Acid Production

In particular embodiments, the methods of the invention are used for thegeneration of genetically engineered micro-organisms capable of theproduction of fatty esters, such as fatty acid methyl esters (“FAME”)and fatty acid ethyl esters (“FAEE”),

Typically, host cells can be engineered to produce fatty esters from acarbon source, such as an alcohol, present in the medium, by expressionor overexpression of a gene encoding a thioesterase, a gene encoding anacyl-CoA synthase, and a gene encoding an ester synthase. Accordingly,the methods provided herein are used to modify a micro-organisms so asto overexpress or introduce a thioesterase gene, a gene encoding anacyl-CoA synthase, and a gene encoding an ester synthase. In particularembodiments, the thioesterase gene is selected from tesA, ‘tesA, tesB,fatB, fatB2, fatB3, fatA1, or fatA. In particular embodiments, the geneencoding an acyl-CoA synthase is selected from fadDJadK, BH3103,pfl-4354, EAV15023, fadD1, fadD2, RPC_4074,fadDD35, fadDD22, faa39, oran identified gene encoding an enzyme having the same properties. Inparticular embodiments, the gene encoding an ester synthase is a geneencoding a synthase/acyl-CoA:diacylglycerol acyltransferase fromSimmondsia chinensis, Acinetobacter sp. ADP, Alcanivorax borkumensis,Pseudomonas aeruginosa, Fundibacter jadensis, Arabidopsis thaliana, orAlcaligenes eutrophus, or a variant thereof. Additionally oralternatively, the methods provided herein are used to decreaseexpression in said micro-organism of at least one of a gene encoding anacyl-CoA dehydrogenase, a gene encoding an outer membrane proteinreceptor, and a gene encoding a transcriptional regulator of fatty acidbiosynthesis. In particular embodiments one or more of these genes isinactivated, such as by introduction of a mutation. In particularembodiments, the gene encoding an acyl-CoA dehydrogenase is fadE. Inparticular embodiments, the gene encoding a transcriptional regulator offatty acid biosynthesis encodes a DNA transcription repressor, forexample, fabR.

Additionally or alternatively, said micro-organism is modified to reduceexpression of at least one of a gene encoding a pyruvate formate lyase,a gene encoding a lactate dehydrogenase, or both. In particularembodiments, the gene encoding a pyruvate formate lyase is pflB. Inparticular embodiments, the gene encoding a lactate dehydrogenase isIdhA. In particular embodiments one or more of these genes isinactivated, such as by introduction of a mutation therein.

In particular embodiments, the micro-organism is selected from the genusEscherichia, Bacillus, Lactobacillus, Rhodococcus, Synechococcus,Synechocystis, Pseudomonas, Aspergillus, Trichoderma, Neurospora,Fusarium, Humicola, Rhizomucor, Kluyveromyces, Pichia, Mucor,Myceliophtora, Penicillium, Phanerochaete, Pleurotus, Trametes,Chrysosporium, Saccharomyces, Stenotrophomonas, Schizosaccharomyces,Yarrowia, or Streptomyces.

The Use of C2c1 or C2c3 in the Generation of Micro-Organisms Capable ofOrganic Acid Production

The methods provided herein are further used to engineer micro-organismscapable of organic acid production, more particularly from pentose orhexose sugars. In particular embodiments, the methods compriseintroducing into a micro-organism an exogenous LDH gene. In particularembodiments, the organic acid production in said micro-organisms isadditionally or alternatively increased by inactivating endogenous genesencoding proteins involved in an endogenous metabolic pathway whichproduces a metabolite other than the organic acid of interest and/orwherein the endogenous metabolic pathway consumes the organic acid. Inparticular embodiments, the modification ensures that the production ofthe metabolite other than the organic acid of interest is reduced.According to particular embodiments, the methods are used to introduceat least one engineered gene deletion and/or inactivation of anendogenous pathway in which the organic acid is consumed or a geneencoding a product involved in an endogenous pathway which produces ametabolite other than the organic acid of interest. In particularembodiments, the at least one engineered gene deletion or inactivationis in one or more gene encoding an enzyme selected from the groupconsisting of pyruvate decarboxylase (pdc), fumarate reductase, alcoholdehydrogenase (adh), acetaldehyde dehydrogenase, phosphoenolpyruvatecarboxylase (ppc), D-lactate dehydrogenase (d-ldh), L-lactatedehydrogenase (1-ldh), lactate 2-monooxygenase. In further embodimentsthe at least one engineered gene deletion and/or inactivation is in anendogenous gene encoding pyruvate decarboxylase (pdc).

In further embodiments, the micro-organism is engineered to producelactic acid and the at least one engineered gene deletion and/orinactivation is in an endogenous gene encoding lactate dehydrogenase.Additionally or alternatively, the micro-organism comprises at least oneengineered gene deletion or inactivation of an endogenous gene encodinga cytochrome-dependent lactate dehydrogenase, such as a cytochromeB2-dependent L-lactate dehydrogenase.

The Use of C2c1 or C2c3 in the Generation of Improved Xylose orCellobiose Utilizing Yeasts Strains

In particular embodiments, the C2c1 or C2c3 CRISPR system may be appliedto select for improved xylose or cellobiose utilizing yeast strains.Error-prone PCR can be used to amplify one (or more) genes involved inthe xylose utilization or cellobiose utilization pathways. Examples ofgenes involved in xylose utilization pathways and cellobiose utilizationpathways may include, without limitation, those described in Ha, S. J.,et al. (2011) Proc. Natl. Acad. Sci. USA 108(2):504-9 and Galazka, J.M., et al. (2010) Science 330(6000):84-6. Resulting libraries ofdouble-stranded DNA molecules, each comprising a random mutation in sucha selected gene could be co-transformed with the components of the C2c1or C2c3 CRISPR system into a yeast strain (for instance S288C) andstrains can be selected with enhanced xylose or cellobiose utilizationcapacity, as described in WO2015138855.

The Use of C2c1 or C293 in the Generation of Improved Yeasts Strains forUse in Isoprenoid Biosynthesis

Tadas Jakočiūnas et al. described the successful application of amultiplex CRISPR/Cas9 system for genome engineering of up to 5 differentgenomic loci in one transformation step in baker's yeast Saccharomycescerevisiae (Metabolic Engineering Volume 28, March 2015, Pages 213-222)resulting in strains with high mevalonate production, a key intermediatefor the industrially important isoprenoid biosynthesis pathway. Inparticular embodiments, the C2c1 or C2c3 CRISPR system may be applied ina multiplex genome engineering method as described herein foridentifying additional high producing yeast strains for use inisoprenoid synthesis.

The Use of C2c1 or C293 in the Generation of Lactic Acid ProducingYeasts Strains

In another embodiment, successful application of a multiplex C2c1 orC2c3 CRISPR system is encompassed. In analogy with Vratislav Stovicek etal. (Metabolic Engineering Communications, Volume 2, December 2015,Pages 13-22), improved lactic acid-producing strains can be designed andobtained in a single transformation event. In a particular embodiment,the C2c1 or C2c3 CRISPR system is used for simultaneously inserting theheterologous lactate dehydrogenase gene and disruption of two endogenousgenes PDC1 and PDC5 genes.

Further Applications of the C2c1 or C293 CRISPR System in Plants

In particular embodiments, the CRISPR system, and preferably the C2c1 orC2c3 CRISPR system described herein, can be used for visualization ofgenetic element dynamics. For example, CRISPR imaging can visualizeeither repetitive or non-repetitive genomic sequences, report telomerelength change and telomere movements and monitor the dynamics of geneloci throughout the cell cycle (Chen et al., Cell, 2013). These methodsmay also be applied to plants.

Other applications of the CRISPR system, and preferably the C2c1 or C2c3CRISPR system described herein, is the targeted gene disruptionpositive-selection screening in vitro and in vivo (Malina et al., Genesand Development, 2013). These methods may also be applied to plants.

In particular embodiments, fusion of inactive C2c1 or C2c3 endonucleaseswith histone-modifying enzymes can introduce custom changes in thecomplex epigenome (Rusk et al., Nature Methods, 2014). These methods mayalso be applied to plants.

In particular embodiments, the CRISPR system, and preferably the C2c1 orC2c3 CRISPR system described herein, can be used to purify a specificportion of the chromatin and identify the associated proteins, thuselucidating their regulatory roles in transcription (Waldrip et al.,Epigenetics, 2014). These methods may also be applied to plants.

In particular embodiments, present invention can be used as a therapyfor virus removal in plant systems as it is able to cleave both viralDNA and RNA. Previous studies in human systems have demonstrated thesuccess of utilizing CRISPR in targeting the single strand RNA virus,hepatitis C (A. Price, et al., Proc. Natl. Acad. Sci, 2015) as well asthe double stranded DNA virus, hepatitis B (V. Ramanan, et al., Sci.Rep, 2015). These methods may also be adapted for using the C2c1 or C2c3CRISPR system in plants.

In particular embodiments, present invention could be used to altergenome complexity. In further particular embodiment, the CRISPR system,and preferably the C2c1 or C2c3 CRISPR system described herein, can beused to disrupt or alter chromosome number and generate haploid plants,which only contain chromosomes from one parent. Such plants can beinduced to undergo chromosome duplication and converted into diploidplants containing only homozygous alleles (Karimi-Ashtiyani et al.,PNAS, 2015; Anton et al., Nucleus, 2014). These methods may also beapplied to plants.

In particular embodiments, the C2c1 or C2c3 CRISPR system describedherein, can be used for self-cleavage. In these embodiments, thepromotor of the C2c1 or C2c3 enzyme and gRNA can be a constitutivepromotor and a second gRNA is introduced in the same transformationcassette, but controlled by an inducible promoter. This second gRNA canbe designated to induce site-specific cleavage in the C2c1 or C2c3 genein order to create a non-functional C2c1 or C2c3. In a furtherparticular embodiment, the second gRNA induces cleavage on both ends ofthe transformation cassette, resulting in the removal of the cassettefrom the host genome. This system offers a controlled duration ofcellular exposure to the Cas enzyme and further minimizes off-targetediting. Furthermore, cleavage of both ends of a CRISPR/Cas cassette canbe used to generate transgene-free TO plants with bi-allelic mutations(as described for Cas9 e.g. Moore et al., Nucleic Acids Research, 2014;Schaeffer et al., Plant Science, 2015). The methods of Moore et al. maybe applied to the C2c1 or C2c3 CRISPR systems described herein.

Sugano et al. (Plant Cell Physiol. 2014 March;55(3):475-81. doi:10.1093/pcp/pcu014. Epub 2014 Jan. 18) reports the application ofCRISPR-Cas9 to targeted mutagenesis in the liverwort Marchantiapolymorpha L., which has emerged as a model species for studying landplant evolution. The U6 promoter of M. polymorpha was identified andcloned to express the gRNA. The target sequence of the gRNA was designedto disrupt the gene encoding auxin response factor 1 (ARF1) in M.polymorpha. Using Agrobacterium-mediated transformation, Sugano et al.isolated stable mutants in the gametophyte generation of M. polymorpha.CRISPR-Cas9-based site-directed mutagenesis in vivo was achieved usingeither the Cauliflower mosaic virus 35S or M. polymorpha EF1α promoterto express Cas9. Isolated mutant individuals showing an auxin-resistantphenotype were not chimeric. Moreover, stable mutants were produced byasexual reproduction of T1 plants. Multiple arfl alleles were easilyestablished using CRIPSR-Cas9-based targeted mutagenesis. The methods ofSugano et al. may be applied to the C2c1 or C2c3 effector protein systemof the present invention.

Kabadi et al. (Nucleic Acids Res. 2014 Oct. 29; 42(19):e147. doi:10.1093/nar/gku749. Epub 2014 Aug. 13) developed a single lentiviralsystem to express a Cas9 variant, a reporter gene and up to four sgRNAsfrom independent RNA polymerase III promoters that are incorporated intothe vector by a convenient Golden Gate cloning method. Each sgRNA wasefficiently expressed and can mediate multiplex gene editing andsustained transcriptional activation in immortalized and primary humancells. The methods of Kabadi et al. may be applied to the C2c1 or C2c3effector protein system of the present invention.

Ling et al. (BMC Plant Biology 2014, 14:327) developed a CRISPR-Cas9binary vector set based on the pGreen or pCAMBIA backbone, as well as agRNA This toolkit requires no restriction enzymes besides BsaI togenerate final constructs harboring maize-codon optimized Cas9 and oneor more gRNAs with high efficiency in as little as one cloning step. Thetoolkit was validated using maize protoplasts, transgenic maize lines,and transgenic Arabidopsis lines and was shown to exhibit highefficiency and specificity. More importantly, using this toolkit,targeted mutations of three Arabidopsis genes were detected intransgenic seedlings of the T1 generation. Moreover, the multiple-genemutations could be inherited by the next generation. (guide RNA)modulevector set, as a toolkit for multiplex genome editing in plants. Thetoolbox of Lin et al. may be applied to the C2c1 or C2c3 effectorprotein system of the present invention.

Protocols for targeted plant genome editing via CRISPR-Cas9 are alsoavailable in volume 1284 of the series Methods in Molecular Biology pp239-255 10 Feb. 2015. A detailed procedure to design, construct, andevaluate dual gRNAs for plant codon optimized Cas9 (pcoCas9) mediatedgenome editing using Arabidopsis thaliana and Nicotiana benthamianaprotoplasts s model cellular systems are described. Strategies to applythe CRISPR-Cas9 system to generating targeted genome modifications inwhole plants are also discussed. The protocols described in the chaptermay be applied to the C2c1 or C2c3 effector protein system of thepresent invention.

Ma et al. (Mol Plant. 2015 Aug. 3; 8(8):1274-84. doi:10.1016/j.molp.2015.04.007) reports robust CRISPR-Cas9 vector system,utilizing a plant codon optimized Cas9 gene, for convenient andhigh-efficiency multiplex genome editing in monocot and dicot plants. Maet al. designed PCR-based procedures to rapidly generate multiple sgRNAexpression cassettes, which can be assembled into the binary CRISPR-Cas9vectors in one round of cloning by Golden Gate ligation or GibsonAssembly. With this system, Ma et al. edited 46 target sites in ricewith an average 85.4% rate of mutation, mostly in biallelic andhomozygous status. Ma et al. provide examples of loss-of-function genemutations in T0 rice and T1 Arabidopsis plants by simultaneous targetingof multiple (up to eight) members of a gene family, multiple genes in abiosynthetic pathway, or multiple sites in a single gene. The methods ofMa et al. may be applied to the C2c1 or C2c3 effector protein system ofthe present invention.

Lowder et al. (Plant Physiol. 2015 Aug. 21. pii: pp. 00636.2015) alsodeveloped a CRISPR-Cas9 toolbox enables multiplex genome editing andtranscriptional regulation of expressed, silenced or non-coding genes inplants. This toolbox provides researchers with a protocol and reagentsto quickly and efficiently assemble functional CRISPR-Cas9 T-DNAconstructs for monocots and dicots using Golden Gate and Gateway cloningmethods. It comes with a full suite of capabilities, includingmultiplexed gene editing and transcriptional activation or repression ofplant endogenous genes. T-DNA based transformation technology isfundamental to modern plant biotechnology, genetics, molecular biologyand physiology. As such, we developed a method for the assembly of Cas9(WT, nickase or dCas9) and gRNA(s) into a T-DNA destination-vector ofinterest. The assembly method is based on both Golden Gate assembly andMultiSite Gateway recombination. Three modules are required forassembly. The first module is a Cas9 entry vector, which containspromoterless Cas9 or its derivative genes flanked by attL1 and attR5sites. The second module is a gRNA entry vector which contains entrygRNA expression cassettes flanked by attL5 and attL2 sites. The thirdmodule includes attR1-attR2-containing destination T-DNA vectors thatprovide promoters of choice for Cas9 expression. The toolbox of Lowderet al. may be applied to the C2c1 or C2c3 effector protein system of thepresent invention.

In an advantageous embodiment, the plant may be a tree. The presentinvention may also utilize the herein disclosed CRISPR Cas system forherbaceous systems (see, e.g., Belhaj et al., Plant Methods 9: 39 andHarrison et al., Genes & Development 28: 1859-1872). In a particularlyadvantageous embodiment, the CRISPR Cas system of the present inventionmay target single nucleotide polymorphisms (SNPs) in trees (see, e.g.,Zhou et al., New Phytologist, Volume 208, Issue 2, pages 298-301,October 2015). In the Zhou et al. study, the authors applied a CRISPRCas system in the woody perennial Populus using the 4-coumarate:CoAligase (4CL) gene family as a case study and achieved 100% mutationalefficiency for two 4CL genes targeted, with every transformant examinedcarrying biallelic modifications. In the Zhou et al., study, theCRISPR-Cas9 system was highly sensitive to single nucleotidepolymorphisms (SNPs), as cleavage for a third 4CL gene was abolished dueto SNPs in the target sequence. These methods may be applied to the C2c1or C2c3 effector protein system of the present invention.

The methods of Zhou et al. (New Phytologist, Volume 208, Issue 2, pages298-301, October 2015) may be applied to the present invention asfollows. Two 4CL genes, 4CL1 and 4CL2, associated with lignin andflavonoid biosynthesis, respectively are targeted for CRISPR-Cas9editing. The Populus tremula x alba clone 717-1B4 routinely used fortransformation is divergent from the genome-sequenced Populustrichocarpa. Therefore, the 4CL1 and 4CL2 gRNAs designed from thereference genome are interrogated with in-house 717 RNA-Seq data toensure the absence of SNPs which could limit Cas efficiency. A thirdgRNA designed for 4CL5, a genome duplicate of 4CL1, is also included.The corresponding 717 sequence harbors one SNP in each allelenear/within the PAM, both of which are expected to abolish targeting bythe 4CL5-gRNA. All three gRNA target sites are located within the firstexon. For 717 transformation, the gRNA is expressed from the MedicagoU6.6 promoter, along with a human codon-optimized Cas under control ofthe CaMV 35S promoter in a binary vector. Transformation with theCas-only vector can serve as a control. Randomly selected 4CL1 and 4CL2lines are subjected to amplicon-sequencing. The data is then processedand biallelic mutations are confirmed in all cases. These methods may beapplied to the C2c1 or C2c3 effector protein system of the presentinvention.

In plants, pathogens are often host-specific. For example, Fusariumoxysporum f. sp. lycopersici causes tomato wilt but attacks only tomato,and F. oxysporum f. dianthus Puccinia graminis f. sp. tritici attacksonly wheat. Plants have existing and induced defenses to resist mostpathogens. Mutations and recombination events across plant generationslead to genetic variability that gives rise to susceptibility,especially as pathogens reproduce with more frequency than plants. Inplants there can be non-host resistance, e.g., the host and pathogen areincompatible. There can also be Horizontal Resistance, e.g., partialresistance against all races of a pathogen, typically controlled by manygenes and Vertical Resistance, e.g., complete resistance to some racesof a pathogen but not to other races, typically controlled by a fewgenes. In a Gene-for-Gene level, plants and pathogens evolve together,and the genetic changes in one balance changes in other. Accordingly,using Natural Variability, breeders combine most useful genes for Yield,Quality, Uniformity, Hardiness, Resistance. The sources of resistancegenes include native or foreign Varieties, Heirloom Varieties, WildPlant Relatives, and Induced Mutations, e.g., treating plant materialwith mutagenic agents. Using the present invention, plant breeders areprovided with a new tool to induce mutations. Accordingly, one skilledin the art can analyze the genome of sources of resistance genes, and inVarieties having desired characteristics or traits employ the presentinvention to induce the rise of resistance genes, with more precisionthan previous mutagenic agents and hence accelerate and improve plantbreeding programs.

Improved Plants and Yeast Cells

The present invention also provides plants and yeast cells obtainableand obtained by the methods provided herein. The improved plantsobtained by the methods described herein may be useful in food or feedproduction through expression of genes which, for instance ensuretolerance to plant pests, herbicides, drought, low or high temperatures,excessive water, etc.

The improved plants obtained by the methods described herein, especiallycrops and algae may be useful in food or feed production throughexpression of, for instance, higher protein, carbohydrate, nutrient orvitamin levels than would normally be seen in the wildtype. In thisregard, improved plants, especially pulses and tubers are preferred.

Improved algae or other plants such as rape may be particularly usefulin the production of vegetable oils or biofuels such as alcohols(especially methanol and ethanol), for instance. These may be engineeredto express or overexpress high levels of oil or alcohols for use in theoil or biofuel industries.

The invention also provides for improved parts of a plant. Plant partsinclude, but are not limited to, leaves, stems, roots, tubers, seeds,endosperm, ovule, and pollen. Plant parts as envisaged herein may beviable, nonviable, regeneratable, and/or non-regeneratable.

It is also encompassed herein to provide plant cells and plantsgenerated according to the methods of the invention. Gametes, seeds,embryos, either zygotic or somatic, progeny or hybrids of plantscomprising the genetic modification, which are produced by traditionalbreeding methods, are also included within the scope of the presentinvention. Such plants may contain a heterologous or foreign DNAsequence inserted at or instead of a target sequence. Alternatively,such plants may contain only an alteration (mutation, deletion,insertion, substitution) in one or more nucleotides. As such, suchplants will only be different from their progenitor plants by thepresence of the particular modification.

Thus, the invention provides a plant, animal or cell, produced by thepresent methods, or a progeny thereof. The progeny may be a clone of theproduced plant or animal, or may result from sexual reproduction bycrossing with other individuals of the same species to introgressfurther desirable traits into their offspring. The cell may be in vivoor ex vivo in the cases of multicellular organisms, particularly animalsor plants.

C2C1 or C2C3 Effector Protein Complexes in Non-Human Organisms/Animals

The present invention may also be extended to other agriculturalapplications such as, for example, farm and production animals. Forexample, pigs have many features that make them attractive as biomedicalmodels, especially in regenerative medicine. In particular, pigs withsevere combined immunodeficiency (SCID) may provide useful models forregenerative medicine, xenotransplantation, and tumor development andwill aid in developing therapies for human SCID patients. Lee et al.,(Proc Natl Acad Sci USA. 2014 May 20; 111(20):7260-5) utilized areporter-guided transcription activator-like effector nuclease (TALEN)system to generated targeted modifications of recombination activatinggene (RAG) 2 in somatic cells at high efficiency, including some thataffected both alleles. The C2c1 or C2c3 effector protein may be appliedto a similar system.

The methods of Lee et al., (Proc Natl Acad Sci USA. 2014 May 20;111(20):7260-5) may be applied to the present invention analogously asfollows. Mutated pigs are produced by targeted modification of RAG2 infetal fibroblast cells followed by SCNT and embryo transfer. Constructscoding for CRISPR Cas and a reporter are electroporated intofetal-derived fibroblast cells. After 48 h, transfected cells expressingthe green fluorescent protein are sorted into individual wells of a96-well plate at an estimated dilution of a single cell per well.Targeted modification of RAG2 are screened by amplifying a genomic DNAfragment flanking any CRISPR Cas cutting sites followed by sequencingthe PCR products. After screening and ensuring lack of off-sitemutations, cells carrying targeted modification of RAG2 are used forSCNT. The polar body, along with a portion of the adjacent cytoplasm ofoocyte, presumably containing the metaphase II plate, are removed, and adonor cell are placed in the perivitelline. The reconstructed embryosare then electrically prorated to fuse the donor cell with the oocyteand then chemically activated. The activated embryos are incubated inPorcine Zygote Medium 3 (PZM3) with 0.5 μM Scriptaid (S7817;Sigma-Aldrich) for 14-16 h. Embryos are then washed to remove theScriptaid and cultured in PZM3 until they were transferred into theoviducts of surrogate pigs.

The present invention is also applicable to modifying SNPs of otheranimals, such as cows. Tan et al. (Proc Natl Acad Sci USA. 2013 Oct. 8;110(41): 16526-16531) expanded the livestock gene editing toolbox toinclude transcription activator-like (TAL) effector nuclease (TALEN)-and clustered regularly interspaced short palindromic repeats(CRISPR)/Cas9-stimulated homology-directed repair (HDR) using plasmid,rAAV, and oligonucleotide templates. Gene specific gRNA sequences werecloned into the Church lab gRNA vector (Addgene ID: 41824) according totheir methods (Mali P, et al. (2013) RNA-Guided Human Genome Engineeringvia Cas9. Science 339(6121):823-826). The Cas9 nuclease was providedeither by co-transfection of the hCas9 plasmid (Addgene ID: 41815) ormRNA synthesized from RCIScript-hCas9. This RCIScript-hCas9 wasconstructed by sub-cloning the XbaI-AgeI fragment from the hCas9 plasmid(encompassing the hCas9 cDNA) into the RCIScript plasmid.

Heo et al. (Stem Cells Dev. 2015 Feb. 1; 24(3):393-402. doi:10.1089/scd.2014.0278. Epub 2014 Nov. 3) reported highly efficient genetargeting in the bovine genome using bovine pluripotent cells andclustered regularly interspaced short palindromic repeat (CRISPR)/Cas9nuclease. First, Heo et al. generate induced pluripotent stem cells(iPSCs) from bovine somatic fibroblasts by the ectopic expression ofyamanaka factors and GSK3β and MEK inhibitor (2i) treatment. Heo et al.observed that these bovine iPSCs are highly similar to naïve pluripotentstem cells with regard to gene expression and developmental potential interatomas. Moreover, CRISPR-Cas9 nuclease, which was specific for thebovine NANOG locus, showed highly efficient editing of the bovine genomein bovine iPSCs and embryos.

Igenity® provides a profile analysis of animals, such as cows, toperform and transmit traits of economic traits of economic importance,such as carcass composition, carcass quality, maternal and reproductivetraits and average daily gain. The analysis of a comprehensive Igenity®profile begins with the discovery of DNA markers (most often singlenucleotide polymorphisms or SNPs). All the markers behind the Igenity®profile were discovered by independent scientists at researchinstitutions, including universities, research organizations, andgovernment entities such as USDA. Markers are then analyzed at Igenity®in validation populations. Igenity® uses multiple resource populationsthat represent various production environments and biological types,often working with industry partners from the seedstock, cow-calf,feedlot and/or packing segments of the beef industry to collectphenotypes that are not commonly available. Cattle genome databases arewidely available, see, e.g., the NAGRP Cattle Genome CoordinationProgram (www.animalgenome.org/cattle/maps/db.html). Thus, the presentinvention maybe applied to target bovine SNPs. One of skill in the artmay utilize the above protocols for targeting SNPs and apply them tobovine SNPs as described, for example, by Tan et al. or Heo et al.

Qingjian Zou et al. (Journal of Molecular Cell Biology, Advance Accesspublished Oct. 12, 2015) demonstrated increased muscle mass in dogs bytargeting the first exon of the dog Myostatin (MSTN) gene (a negativeregulator of skeletal muscle mass). First, the efficiency of the sgRNAwas validated, using cotransfection of the sgRNA targeting MSTN with aCas9 vector into canine embryonic fibroblasts (CEFs). Thereafter, MSTNKO dogs were generated by micro-injecting embryos with normal morphologywith a mixture of Cas9 mRNA and MSTN sgRNA and auto-transplantation ofthe zygotes into the oviduct of the same female dog. The knock-outpuppies displayed an obvious muscular phenotype on thighs compared withits wild-type littermate sister. This can also be performed using theC2c1 or C2c3 CRISPR systems provided herein.

Livestock—Pigs

Viral targets in livestock may include, in some embodiments, porcineCD163, for example on porcine macrophages. CD163 is associated withinfection (thought to be through viral cell entry) by PRRSv (PorcineReproductive and Respiratory Syndrome virus, an arterivirus). Infectionby PRRSv, especially of porcine alveolar macrophages (found in thelung), results in a previously incurable porcine syndrome (“Mysteryswine disease” or “blue ear disease”) that causes suffering, includingreproductive failure, weight loss and high mortality rates in domesticpigs. Opportunistic infections, such as enzootic pneumonia, meningitisand ear oedema, are often seen due to immune deficiency through loss ofmacrophage activity. It also has significant economic and environmentalrepercussions due to increased antibiotic use and financial loss (anestimated $660m per year).

As reported by Kristin M Whitworth and Dr Randall Prather et al. (NatureBiotech 3434 published online 7 Dec. 2015) at the University of Missouriand in collaboration with Genus Plc, CD163 was targeted usingCRISPR-Cas9 and the offspring of edited pigs were resistant when exposedto PRRSv. One founder male and one founder female, both of whom hadmutations in exon 7 of CD163, were bred to produce offspring. Thefounder male possessed an 11-bp deletion in exon 7 on one allele, whichresults in a frameshift mutation and missense translation at amino acid45 in domain 5 and a subsequent premature stop codon at amino acid 64.The other allele had a 2-bp addition in exon 7 and a 377-bp deletion inthe preceding intron, which were predicted to result in the expressionof the first 49 amino acids of domain 5, followed by a premature stopcode at amino acid 85. The sow had a 7 bp addition in one allele thatwhen translated was predicted to express the first 48 amino acids ofdomain 5, followed by a premature stop codon at amino acid 70. The sow'sother allele was unamplifiable. Selected offspring were predicted to bea null animal (CD163−/−), i.e. a CD163 knock out.

Accordingly, in some embodiments, porcine alveolar macrophages may betargeted by the CRISPR protein. In some embodiments, porcine CD163 maybe targeted by the CRISPR protein. In some embodiments, porcine CD163may be knocked out through induction of a DSB or through insertions ordeletions, for example targeting deletion or modification of exon 7,including one or more of those described above, or in other regions ofthe gene, for example deletion or modification of exon 5.

An edited pig and its progeny are also envisaged, for example a CD163knock out pig. This may be for livestock, breeding or modelling purposes(i.e. a porcine model). Semen comprising the gene knock out is alsoprovided.

CD163 is a member of the scavenger receptor cysteine-rich (SRCR)superfamily. Based on in vitro studies SRCR domain 5 of the protein isthe domain responsible for unpackaging and release of the viral genome.As such, other members of the SRCR superfamily may also be targeted inorder to assess resistance to other viruses. PRRSV is also a member ofthe mammalian arterivirus group, which also includes murine lactatedehydrogenase-elevating virus, simian hemorrhagic fever virus and equinearteritis virus. The arteriviruses share important pathogenesisproperties, including macrophage tropism and the capacity to cause bothsevere disease and persistent infection. Accordingly, arteriviruses, andin particular murine lactate dehydrogenase-elevating virus, simianhemorrhagic fever virus and equine arteritis virus, may be targeted, forexample through porcine CD163 or homologues thereof in other species,and murine, simian and equine models and knockout also provided.

Indeed, this approach may be extended to viruses or bacteria that causeother livestock diseases that may be transmitted to humans, such asSwine Influenza Virus (SIV) strains which include influenza C and thesubtypes of influenza A known as H1N1, H1N2, H2N1, H3N1, H3N2, and H2N3,as well as pneumonia, meningitis and oedema mentioned above.

Therapeutic Targeting with RNA-Guided C2c1 or C2C3 Effector ProteinComplex

As will be apparent, it is envisaged that the present system can be usedto target any polynucleotide sequence of interest. The inventionprovides a non-naturally occurring or engineered composition, or one ormore polynucleotides encoding components of said composition, or vectoror delivery systems comprising one or more polynucleotides encodingcomponents of said composition for use in a modifying a target cell invivo, ex vivo or in vitro and, may be conducted in a manner alters thecell such that once modified the progeny or cell line of the CRISPRmodified cell retains the altered phenotype. The modified cells andprogeny may be part of a multi-cellular organism such as a plant oranimal with ex vivo or in vivo application of CRISPR system to desiredcell types. The CRISPR invention may be a therapeutic method oftreatment. The therapeutic method of treatment may comprise gene orgenome editing, or gene therapy.

Treating Pathogens, Like Bacterial, Fungal and Parasitic Pathogens

The present invention may also be applied to treat bacterial, fungal andparasitic pathogens. Most research efforts have focused on developingnew antibiotics, which once developed, would nevertheless be subject tothe same problems of drug resistance. The invention provides novelCRISPR-based alternatives which overcome those difficulties.Furthermore, unlike existing antibiotics, CRISPR-based treatments can bemade pathogen specific, inducing bacterial cell death of a targetpathogen while avoiding beneficial bacteria.

Jiang et al. (“RNA-guided editing of bacterial genomes using CRISPR-Cassystems,” Nature Biotechnology vol. 31, p. 233-9, March 2013) used aCRISPR-Cas9 system to mutate or kill S. pneumoniae and E. coli. Thework, which introduced precise mutations into the genomes, relied ondual-RNA:Cas9-directed cleavage at the targeted genomic site to killunmutated cells and circumvented the need for selectable markers orcounter-selection systems. CRISPR systems have be used to reverseantibiotic resistance and eliminate the transfer of resistance betweenstrains. Bickard et al. showed that Cas9, reprogrammed to targetvirulence genes, kills virulent, but not avirulent, S. aureus.Reprogramming the nuclease to target antibiotic resistance genesdestroyed staphylococcal plasmids that harbor antibiotic resistancegenes and immunized against the spread of plasmid-bome resistance genes.(see, Bikard et al., “Exploiting CRISPR-Cas nucleases to producesequence-specific antimicrobials,” Nature Biotechnology vol. 32,1146-1150, doi:10.1038/nbt.3043, published online 5 Oct. 2014.) Bikardshowed that CRISPR-Cas9 antimicrobials function in vivo to kill S.aureus in a mouse skin colonization model. Similarly, Yosef et al used aCRISPR system to target genes encoding enzymes that confer resistance toβ-lactam antibiotics (see Yousef et al., “Temperate and lyticbacteriophages programmed to sensitize and kill antibiotic-resistantbacteria,” Proc. Nat. Acad. Sci. USA, vol. 112, p. 7267-7272, doi:10.1073/pnas.1500107112 published online May 18, 2015).

CRISPR systems can be used to edit genomes of parasites that areresistant to other genetic approaches. For example, a CRISPR-Cas9 systemwas shown to introduce double-stranded breaks into the in the Plasmodiumyoelii genome (see, Zhang et al., “Efficient Editing of Malaria ParasiteGenome Using the CRISPR/Cas9 System,” mBio. vol. 5, e01414-14,July-August 2014). Ghorbal et al. (“Genome editing in the human malariaparasite Plasmodium falciparum: using the CRISPR-Cas9 system,” NatureBiotechnology, vol. 32, p. 819-821, doi: 10.1038/nbt.2925, publishedonline Jun. 1, 2014) modified the sequences of two genes, orc1 andkelch13, which have putative roles in gene silencing and emergingresistance to artemisinin, respectively. Parasites that were altered atthe appropriate sites were recovered with very high efficiency, despitethere being no direct selection for the modification, indicating thatneutral or even deleterious mutations can be generated using thissystem. CRISPR-Cas9 is also used to modify the genomes of otherpathogenic parasites, including Toxoplasma gondii (see Shen et al.,“Efficient gene disruption in diverse strains of Toxoplasma gondii usingCRISPR/CAS9,” mBio vol. 5:e01114-14, 2014; and Sidik et al., “EfficientGenome Engineering of Toxoplasma gondii Using CRISPR/Cas9,” PLoS Onevol. 9, e100450, doi: 10.1371/journal.pone.0100450, published onlineJun. 27, 2014).

Vyas et al. (“A Candida albicans CRISPR system permits geneticengineering of essential genes and gene families,” Science Advances,vol. 1, e1500248, DOI: 10.1126/sciadv.1500248, Apr. 3, 2015) employed aCRISPR system to overcome long-standing obstacles to genetic engineeringin C. albicans and efficiently mutate in a single experiment both copiesof several different genes. In an organism where several mechanismscontribute to drug resistance, Vyas produced homozygous double mutantsthat no longer displayed the hyper-resistance to fluconazole orcycloheximide displayed by the parental clinical isolate Can90. Vyasalso obtained homozygous loss-of-function mutations in essential genesof C. albicans by creating conditional alleles. Null alleles of DCR1,which is required for ribosomal RNA processing, are lethal at lowtemperature but viable at high temperature. Vyas used a repair templatethat introduced a nonsense mutation and isolated dcr1,der1 mutants thatfailed to grow at 16° C.

The CRISPR system of the present invention for use in P. falciparum bydisrupting chromosomal loci. Ghorbal et al. (“Genome editing in thehuman malaria parasite Plasmodium falciparum using the CRISPR-Cas9system”, Nature Biotechnology, 32, 819-821 (2014), DOI:10.1038/nbt.2925, Jun. 1, 2014) employed a CRISPR system to introducespecific gene knockouts and single-nucleotide substitutions in themalaria genome. To adapt the CRISPR-Cas9 system to P. falciparum,Ghorbal et al. generated expression vectors for under the control ofplasmodial regulatory elements in the pUF1-Cas9 episome that alsocarries the drug-selectable marker ydhodh, which gives resistance toDSM1, a P. falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitorand for transcription of the sgRNA, used P. falciparum U6 small nuclear(sn)RNA regulatory elements placing the guide RNA and the donor DNAtemplate for homologous recombination repair on the same plasmid, pL7.See also, Zhang C. et al. (“Efficient editing of malaria parasite genomeusing the CRISPR/Cas9 system”, MBio, 2014 Jul. 1; 5(4):E01414-14, doi:10. 1128/MbIO.01414-14) and Wagner et al. (“EfficientCRISPR-Cas9-mediated genome editing in Plasmodium falciparum, NatureMethods 11, 915-918 (2014), DOI: 10.1038/nmeth.3063).

Treating Pathogens, Like Viral Pathogens Such as HIV

Cas-mediated genome editing might be used to introduce protectivemutations in somatic tissues to combat nongenetic or complex diseases.For example, NHEJ-mediated inactivation of the CCR5 receptor inlymphocytes (Lombardo et al., Nat Biotechnol. 2007 November;25(11):1298-306) may be a viable strategy for circumventing HIVinfection, whereas deletion of PCSK9 (Cohen et al., Nat Genet. 2005February; 37(2):161-5) angiopoietin (Musunuru et al., N Engl J Med. 2010Dec. 2; 363(23):2220-7) may provide therapeutic effects againststatin-resistant hypercholesterolemia or hyperlipidemia. Although thesetargets may be also addressed using siRNA-mediated protein knockdown, aunique advantage of NHEJ-mediated gene inactivation is the ability toachieve permanent therapeutic benefit without the need for continuingtreatment. As with all gene therapies, it will of course be important toestablish that each proposed therapeutic use has a favorablebenefit-risk ratio.

Hydrodynamic delivery of plasmid DNA encoding Cas9 nd guide RNA alongwith a repair template into the liver of an adult mouse model oftyrosinemia was shown to be able to correct the mutant Fah gene andrescue expression of the wild-type Fah protein in ˜1 out of 250 cells(Nat Biotechnol. 2014 June; 32(6):551-3). In addition, clinical trialssuccessfully used ZF nucleases to combat HIV infection by ex vivoknockout of the CCR5 receptor. In all patients, HIV DNA levelsdecreased, and in one out of four patients, HIV RNA became undetectable(Tebas et al., N Engl J Med. 2014 Mar. 6; 370(10):901-10). Both of theseresults demonstrate the promise of programmable nucleases as a newtherapeutic platform.

In another embodiment, self-inactivating lentiviral vectors with ansiRNA targeting a common exon shared by HIV tat/rev, anucleolar-localizing TAR decoy, and an anti-CCR5-specific hammerheadribozyme (see, e.g., DiGiusto et al. (2010) Sci Transl Med 2:36ra43) maybe used and/or adapted to the CRISPR-Cas system of the presentinvention. A minimum of 2.5×10⁶ CD34+ cells per kilogram patient weightmay be collected and prestimulated for 16 to 20 hours in X-VIVO 15medium (Lonza) containing 2 μmol/L-glutamine, stem cell factor (100ng/ml), Flt-3 ligand (Flt-3L) (100 ng/ml), and thrombopoietin (10 ng/ml)(CellGenix) at a density of 2×10⁶ cells/ml. Prestimulated cells may betransduced with lentiviral at a multiplicity of infection of 5 for 16 to24 hours in 75-cm² tissue culture flasks coated with fibronectin (25mg/cm²) (RetroNectin, Takara Bio Inc.).

With the knowledge in the art and the teachings in this disclosure theskilled person can correct HSCs as to immunodeficiency condition such asHIV/AIDS comprising contacting an HSC with a CRISPR-Cas9 system thattargets and knocks out CCR5. An guide RNA (and advantageously a dualguide approach, e.g., a pair of different guide RNAs; for instance,guide RNAs targeting of two clinically relevant genes, B2M and CCR5, inprimary human CD4+ T cells and CD34+hematopoietic stem and progenitorcells (HSPCs)) that targets and knocks out CCR5-and-C2c1 or -C2c3protein containing particle is contacted with HSCs. The so contactedcells can be administered; and optionally treated/expanded; cf. Cartier.See also Kiem, “Hematopoietic stem cell-based gene therapy for HIVdisease,” Cell Stem Cell. Feb. 3, 2012; 10(2): 137-147; incorporatedherein by reference along with the documents it cites; Mandal et al,“Efficient Ablation of Genes in Human Hematopoietic Stem and EffectorCells using CRISPR/Cas9,” Cell Stem Cell, Volume 15, Issue 5, p643-652,6 Nov. 2014; incorporated herein by reference along with the documentsit cites. Mention is also made of Ebina, “CRISPR/Cas9 system to suppressHIV-1 expression by editing HIV-1 integrated proviral DNA” SCIENTIFICREPORTS|3: 2510|DOI: 10.1038/srep02510, incorporated herein by referencealong with the documents it cites, as another means for combattingHIV/AIDS using a CRISPR-C2c1 or -C2c3 system.

The rationale for genome editing for HIV treatment originates from theobservation that individuals homozygous for loss of function mutationsin CCR5, a cellular co-receptor for the virus, are highly resistant toinfection and otherwise healthy, suggesting that mimicking this mutationwith genome editing could be a safe and effective therapeutic strategy[Liu, R., et al. Cell 86, 367-377 (1996)]. This idea was clinicallyvalidated when an HIV infected patient was given an allogeneic bonemarrow transplant from a donor homozygous for a loss of function CCR5mutation, resulting in undetectable levels of HIV and restoration ofnormal CD4 T-cell counts [Hutter, G., et al. The New England Journal OfMedicine 360, 692-698 (2009)]. Although bone marrow transplantation isnot a realistic treatment strategy for most HIV patients, due to costand potential graft vs. host disease, HIV therapies that convert apatient's own T-cells into CCR5 are desirable.

Early studies using ZFNs and NHEJ to knockout CCR5 in humanized mousemodels of HIV showed that transplantation of CCR5 edited CD4 T cellsimproved viral load and CD4 T-cell counts [Perez, E. E., et al. NatureBiotechnology 26, 808-816 (2008)]. Importantly, these models also showedthat HIV infection resulted in selection for CCR5 null cells, suggestingthat editing confers a fitness advantage and potentially allowing asmall number of edited cells to create a therapeutic effect.

As a result of this and other promising preclinical studies, genomeediting therapy that knocks out CCR5 in patient T cells has now beentested in humans [Holt, N., et al. Nature Biotechnology 28, 839-847(2010); Li, L., et al. Molecular therapy: the journal of the AmericanSociety of Gene Therapy 21, 1259-1269 (2013)]. In a recent phase Iclinical trial, CD4+ T cells from patients with HIV were removed, editedwith ZFNs designed to knockout the CCR5 gene, and autologouslytransplanted back into patients [Tebas, P., et al. The New EnglandJournal Of Medicine 370, 901-910 (2014)].

In another study (Mandal et al., Cell Stem Cell, Volume 15, Issue 5,p643-652, 6 Nov. 2014), CRISPR-Cas9 has targeted two clinical relevantgenes, B2M and CCR5, in human CD4+ T cells and CD34+ hematopoietic stemand progenitor cells (HSPCs). Use of single RNA guides led to highlyefficient mutagenesis in HSPCs but not in T cells. A dual guide approachimproved gene deletion efficacy in both cell types. HSPCs that hadundergone genome editing with CRISPR-Cas9 retained multilineagepotential. Predicted on- and off-target mutations were examined viatarget capture sequencing in HSPCs and low levels of off-targetmutagenesis were observed at only one site. These results demonstratethat CRISPR-Cas9 can efficiently ablate genes in HSPCs with minimaloff-target mutagenesis, which have broad applicability for hematopoieticcell-based therapy.

Wang et al. (PLoS One. 2014 Dec. 26; 9(12):e115987. doi:10.1371/journal.pone.0115987) silenced CCR5 via CRISPR associatedprotein 9 (Cas9) and single guided RNAs (guide RNAs) with lentiviralvectors expressing Cas9 and CCR5 guide RNAs. Wang et al. showed that asingle round transduction of lentiviral vectors expressing Cas9 and CCR5guide RNAs into HIV-1 susceptible human CD4+ cells yields highfrequencies of CCR5 gene disruption. CCR5 gene-disrupted cells are notonly resistant to R5-tropic HIV-1, including transmitted/founder (T/F)HIV-1 isolates, but also have selective advantage over CCR5gene-undisrupted cells during R5-tropic HIV-1 infection. Genomemutations at potential off-target sites that are highly homologous tothese CCR5 guide RNAs in stably transduced cells even at 84 days posttransduction were not detected by a T7 endonuclease I assay.

Fine et al. (Sci Rep. 2015 Jul. 1; 5:10777. doi: 10.1038/srep10777)identified a two-cassette system expressing pieces of the S. pyogenesCas9 (SpCas9) protein which splice together in cellula to form afunctional protein capable of site-specific DNA cleavage. With specificCRISPR guide strands, Fine et al. demonstrated the efficacy of thissystem in cleaving the HBB and CCR5 genes in human HEK-293T cells as asingle Cas9 and as a pair of Cas9 nickases. The trans-spliced SpCas9(tsSpCas9) displayed ˜35% of the nuclease activity compared with thewild-type SpCas9 (wtSpCas9) at standard transfection doses, but hadsubstantially decreased activity at lower dosing levels. The greatlyreduced open reading frame length of the tsSpCas9 relative to wtSpCas9potentially allows for more complex and longer genetic elements to bepackaged into an AAV vector including tissue-specific promoters,multiplexed guide RNA expression, and effector domain fusions to SpCas9.

Li et al. (J Gen Virol. 2015 August; 96(8):2381-93. doi:10.1099/vir.0.000139. Epub 2015 Apr. 8) demonstrated that CRISPR-Cas9can efficiently mediate the editing of the CCR5 locus in cell lines,resulting in the knockout of CCR5 expression on the cell surface.Next-generation sequencing revealed that various mutations wereintroduced around the predicted cleavage site of CCR5. For each of thethree most effective guide RNAs that were analyzed, no significantoff-target effects were detected at the 15 top-scoring potential sites.By constructing chimeric Ad5F35 adenoviruses carrying CRISPR-Cas9components, Li et al. efficiently transduced primary CD4+T-lymphocytesand disrupted CCR5 expression, and the positively transduced cells wereconferred with HIV-1 resistance.

One of skill in the art may utilize the above studies of, for example,Holt, N., et al. Nature biotechnology 28, 839-847 (2010), Li, L., et al.Molecular therapy: the journal of the American Society of Gene Therapy21, 1259-1269 (2013), Mandal et al., Cell Stem Cell, Volume 15, Issue 5,p643-652, 6 Nov. 2014, Wang et al. (PLoS One. 2014 Dec. 26;9(12):e115987. doi: 10.1371/journal.pone.0115987), Fine et al. (Sci Rep.2015 Jul. 1; 5:10777. doi: 10.1038/srep10777) and Li et al. (J GenVirol. 2015 August; 96(8):2381-93. doi: 10.1099/vir.0.000139. Epub 2015Apr. 8) for targeting CCR5 with the CRISPR Cas system of the presentinvention.

Treating Pathogens, Like Viral Pathogens, Such as HBV

The present invention may also be applied to treat hepatitis B virus(HBV). However, the CRISPR Cas system must be adapted to avoid theshortcomings of RNAi, such as the risk of oversaturating endogenoussmall RNA pathways, by for example, optimizing dose and sequence (see,e.g., Grimm et al., Nature vol. 441, 26 May 2006). For example, lowdoses, such as about 1-10×10¹⁴ particles per human are contemplated. Inanother embodiment, the CRISPR Cas system directed against HBV may beadministered in liposomes, such as a stable nucleic-acid-lipid particle(SNALP) (see, e.g., Morrissey et al., Nature Biotechnology, Vol. 23, No.8, August 2005). Daily intravenous injections of about 1, 3 or 5mg/kg/day of CRISPR Cas targeted to HBV RNA in a SNALP are contemplated.The daily treatment may be over about three days and then weekly forabout five weeks. In another embodiment, the system of Chen et al. (GeneTherapy (2007) 14, 11-19) may be used and/or adapted for the CRISPR Cassystem of the present invention. Chen et al. use a double-strandedadeno-associated virus 8-pseudotyped vector (dsAAV2/8) to deliver shRNA.A single administration of dsAAV2/8 vector (1×10¹² vector genomes permouse), carrying HBV-specific shRNA, effectively suppressed the steadylevel of HBV protein, mRNA and replicative DNA in liver of HBVtransgenic mice, leading to up to 2-3 log₁₀ decrease in HBV load in thecirculation. Significant HBV suppression sustained for at least 120 daysafter vector administration. The therapeutic effect of shRNA was targetsequence dependent and did not involve activation of interferon. For thepresent invention, a CRISPR Cas system directed to HBV may be clonedinto an AAV vector, such as a dsAAV2/8 vector and administered to ahuman, for example, at a dosage of about 1×10¹⁵ vector genomes to about1×10¹⁶ vector genomes per human. In another embodiment, the method ofWooddell et al. (Molecular Therapy vol. 21 no. 5, 973-985 May 2013) maybe used and/or adapted to the CRISPR Cas system of the presentinvention. Woodell et al. show that simple coinjection of ahepatocyte-targeted, N-acetylgalactosamine-conjugated melittin-likepeptide (NAG-MLP) with a liver-tropic cholesterol-conjugated siRNA(chol-siRNA) targeting coagulation factor VII (F7) results in efficientF7 knockdown in mice and nonhuman primates without changes in clinicalchemistry or induction of cytokines. Using transient and transgenicmouse models of HBV infection, Wooddell et al. show that a singlecoinjection of NAG-MLP with potent chol-siRNAs targeting conserved HBVsequences resulted in multilog repression of viral RNA, proteins, andviral DNA with long duration of effect. Intravenous injections, forexample, of about 6 mg/kg of NAG-MLP and 6 mg/kg of HBV specific CRISPRCas may be envisioned for the present invention. In the alternative,about 3 mg/kg of NAG-MLP and 3 mg/kg of HBV specific CRISPR Cas may bedelivered on day one, followed by administration of about 2-3 mg/kg ofNAG-MLP and 2-3 mg/kg of HBV specific CRISPR Cas two weeks later.

Lin et al. (Mol Ther Nucleic Acids. 2014 Aug. 19; 3:e186. doi:10.1038/mtna.2014.38) designed eight gRNAs against HBV of genotype A.With the HBV-specific gRNAs, the CRISPR-Cas9 system significantlyreduced the production of HBV core and surface proteins in Huh-7 cellstransfected with an HBV-expression vector. Among eight screened gRNAs,two effective ones were identified. One gRNA targeting the conserved HBVsequence acted against different genotypes. Using a hydrodynamics-HBVpersistence mouse model, Lin et al. further demonstrated that thissystem could cleave the intrahepatic HBV genome-containing plasmid andfacilitate its clearance in vivo, resulting in reduction of serumsurface antigen levels. These data suggest that the CRISPR-Cas9 systemcould disrupt the HBV-expressing templates both in vitro and in vivo,indicating its potential in eradicating persistent HBV infection.

Dong et al. (Antiviral Res. 2015 June; 118:110-7. doi:10.1016/j.antiviral.2015.03.015. Epub 2015 Apr. 3) used the CRISPR-Cas9system to target the HBV genome and efficiently inhibit HBV infection.Dong et al. synthesized four single-guide RNAs (guide RNAs) targetingthe conserved regions of HBV. The expression of these guide RNAS withCas9 reduced the viral production in Huh7 cells as well as inHBV-replication cell HepG2.2.15. Dong et al. further demonstrated thatCRISPR-Cas9 direct cleavage and cleavage-mediated mutagenesis occurredin HBV cccDNA of transfected cells. In the mouse model carrying HBVcccDNA, injection of guide RNA-Cas9 plasmids via rapid tail veinresulted in the low level of cccDNA and HBV protein.

Liu et al. (J Gen Virol. 2015 August; 96(8):2252-61. doi:10.1099/vir.0.000159. Epub 2015 Apr. 22) designed eight guide RNAs(gRNAs) that targeted the conserved regions of different HBV genotypes,which could significantly inhibit HBV replication both in vitro and invivo to investigate the possibility of using the CRISPR-Cas9 system todisrupt the HBV DNA templates. The HBV-specific gRNA/C2c1 or /C2c3system could inhibit the replication of HBV of different genotypes incells, and the viral DNA was significantly reduced by a single gRNA/C2c1or /C2c3 system and cleared by a combination of different gRNA/C2c1 or/C2c3 systems.

Wang et al. (World J Gastroenterol. 2015 Aug. 28; 21(32):9554-65. doi:10.3748/wjg.v21.i32.9554) designed 15 gRNAs against HBV of genotypesA-D. Eleven combinations of two above gRNAs (dual-gRNAs) covering theregulatory region of HBV were chosen. The efficiency of each gRNA and 11dual-gRNAs on the suppression of HBV (genotypes A-D) replication wasexamined by the measurement of HBV surface antigen (HBsAg) or e antigen(HBeAg) in the culture supernatant. The destruction of HBV-expressingvector was examined in HuH7 cells co-transfected with dual-gRNAs andHBV-expressing vector using polymerase chain reaction (PCR) andsequencing method, and the destruction of cccDNA was examined in HepAD38cells using KCl precipitation, plasmid-safe ATP-dependent DNase (PSAD)digestion, rolling circle amplification and quantitative PCR combinedmethod. The cytotoxicity of these gRNAs was assessed by a mitochondrialtetrazolium assay. All of gRNAs could significantly reduce HBsAg orHBeAg production in the culture supernatant, which was dependent on theregion in which gRNA against. All of dual gRNAs could efficientlysuppress HBsAg and/or HBeAg production for HBV of genotypes A-D, and theefficacy of dual gRNAs in suppressing HBsAg and/or HBeAg production wassignificantly increased when compared to the single gRNA used alone.Furthermore, by PCR direct sequencing we confirmed that these dual gRNAscould specifically destroy HBV expressing template by removing thefragment between the cleavage sites of the two used gRNAs. Mostimportantly, gRNA-5 and gRNA-12 combination not only could efficientlysuppressing HBsAg and/or HBeAg production, but also destroy the cccDNAreservoirs in HepAD38 cells.

Karimova et al. (Sci Rep. 2015 Sep. 3; 5:13734. doi: 10.1038/srep13734)identified cross-genotype conserved HBV sequences in the S and X regionof the HBV genome that were targeted for specific and effective cleavageby a Cas9 nickase. This approach disrupted not only episomal cccDNA andchromosomally integrated HBV target sites in reporter cell lines, butalso HBV replication in chronically and de novo infected hepatoma celllines.

One of skill in the art may utilize the above studies of, for example,Lin et al. (Mol Ther Nucleic Acids. 2014 Aug. 19; 3:e186. doi:10.1038/mtna.2014.38), Dong et al. (Antiviral Res. 2015 June; 118:110-7.doi: 10.1016/j.antiviral.2015.03.015. Epub 2015 Apr. 3), Liu et al. (JGen Virol. 2015 August; 96(8):2252-61. doi: 10.1099/vir.0.000159. Epub2015 Apr. 22), Wang et al. (World J Gastroenterol. 2015 Aug. 28;21(32):9554-65. doi: 10.3748/wjg.v21.i32.9554) and Karimova et al. (SciRep. 2015 Sep. 3; 5:13734. doi: 10.1038/srep13734) for targeting HBVwith the CRISPR Cas system of the present invention.

Chronic hepatitis B virus (HBV) infection is prevalent, deadly, andseldom cured due to the persistence of viral episomal DNA (cccDNA) ininfected cells. Ramanan et al. (Ramanan V, Shlomai A, Cox D B, SchwartzR E, Michailidis E, Bhatta A, Scott D A, Zhang F, Rice C M, Bhatia S N,Sci. Rep. 2015 Jun. 2; 5:10833. doi: 10.1038/srep10833, published online2nd June 2015.) showed that the CRISPR/Cas9 system can specificallytarget and cleave conserved regions in the HBV genome, resulting inrobust suppression of viral gene expression and replication. Uponsustained expression of Cas9 and appropriately chosen guide RNAs, theydemonstrated cleavage of cccDNA by Cas9 and a dramatic reduction in bothcccDNA and other parameters of viral gene expression and replication.Thus, they showed that directly targeting viral episomal DNA is a noveltherapeutic approach to control the virus and possibly cure patients.This is also described in WO2015089465 A1, in the name of the BroadInstitute et al., the contents of which are hereby incorporated byreference.

As such targeting viral episomal DNA in HBV is preferred in someembodiments.

The present invention may also be applied to treat pathogens, e.g.bacterial, fungal and parasitic pathogens. Most research efforts havefocused on developing new antibiotics, which once developed, wouldnevertheless be subject to the same problems of drug resistance. Theinvention provides novel CRISPR-based alternatives which overcome thosedifficulties. Furthermore, unlike existing antibiotics, CRISPR-basedtreatments can be made pathogen specific, inducing bacterial cell deathof a target pathogen while avoiding beneficial bacteria.

The present invention may also be applied to treat hepatitis C virus(HCV). The methods of Roelvinki et al. (Molecular Therapy vol. 20 no. 9,1737-1749 September 2012) may be applied to the CRISPR Cas system. Forexample, an AAV vector such as AAV8 may be a contemplated vector and forexample a dosage of about 1.25×1011 to 1.25×1013 vector genomes perkilogram body weight (vg/kg) may be contemplated. The present inventionmay also be applied to treat pathogens, e.g. bacterial, fungal andparasitic pathogens. Most research efforts have focused on developingnew antibiotics, which once developed, would nevertheless be subject tothe same problems of drug resistance. The invention provides novelCRISPR-based alternatives which overcome those difficulties.Furthermore, unlike existing antibiotics, CRISPR-based treatments can bemade pathogen specific, inducing bacterial cell death of a targetpathogen while avoiding beneficial bacteria.

Jiang et al. (“RNA-guided editing of bacterial genomes using CRISPR-Cassystems,” Nature Biotechnology vol. 31, p. 233-9, March 2013) used aCRISPR-Cas9 system to mutate or kill S. pneumoniae and E. coli. Thework, which introduced precise mutations into the genomes, relied ondual-RNA:Cas9-directed cleavage at the targeted genomic site to killunmutated cells and circumvented the need for selectable markers orcounter-selection systems. CRISPR systems have be used to reverseantibiotic resistance and eliminate the transfer of resistance betweenstrains. Bickard et al. showed that Cas9, reprogrammed to targetvirulence genes, kills virulent, but not avirulent, S. aureus.Reprogramming the nuclease to target antibiotic resistance genesdestroyed staphylococcal plasmids that harbor antibiotic resistancegenes and immunized against the spread of plasmid-bome resistance genes.(see, Bikard et al., “Exploiting CRISPR-Cas nucleases to producesequence-specific antimicrobials,” Nature Biotechnology vol. 32,1146-1150, doi:10.1038/nbt.3043, published online 5 Oct. 2014.) Bikardshowed that CRISPR-Cas9 antimicrobials function in vivo to kill S.aureus in a mouse skin colonization model. Similarly, Yosef et al used aCRISPR system to target genes encoding enzymes that confer resistance toβ-lactam antibiotics (see Yousef et al., “Temperate and lyticbacteriophages programmed to sensitize and kill antibiotic-resistantbacteria,” Proc. Natl. Acad. Sci. USA, vol. 112, p. 7267-7272, doi:10.1073/pnas.1500107112 published online May 18, 2015).

CRISPR systems can be used to edit genomes of parasites that areresistant to other genetic approaches. For example, a CRISPR-Cas9 systemwas shown to introduce double-stranded breaks into the in the Plasmodiumyoelii genome (see, Zhang et al., “Efficient Editing of Malaria ParasiteGenome Using the CRISPR/Cas9 System,” mBio. vol. 5, e01414-14,July-August 2014). Ghorbal et al. (“Genome editing in the human malariaparasite Plasmodium falciparum using the CRISPR-Cas9 system,” NatureBiotechnology, vol. 32, p. 819-821, doi: 10.1038/nbt.2925, publishedonline Jun. 1, 2014) modified the sequences of two genes, orc1 andkelch13, which have putative roles in gene silencing and emergingresistance to artemisinin, respectively. Parasites that were altered atthe appropriate sites were recovered with very high efficiency, despitethere being no direct selection for the modification, indicating thatneutral or even deleterious mutations can be generated using thissystem. CRISPR-Cas9 is also used to modify the genomes of otherpathogenic parasites, including Toxoplasma gondii (see Shen et al.,“Efficient gene disruption in diverse strains of Toxoplasma gondii usingCRISPR/CAS9,” mBio vol. 5:e01114-14, 2014; and Sidik et al., “EfficientGenome Engineering of Toxoplasma gondii Using CRISPR/Cas9,” PLoS Onevol. 9, e100450, doi: 10.1371/journal.pone.0100450, published onlineJun. 27, 2014).

Vyas et al. (“A Candida albicans CRISPR system permits geneticengineering of essential genes and gene families,” Science Advances,vol. 1, e1500248, DOI: 10.1126/sciadv.1500248, Apr. 3, 2015) employed aCRISPR system to overcome long-standing obstacles to genetic engineeringin C. albicans and efficiently mutate in a single experiment both copiesof several different genes. In an organism where several mechanismscontribute to drug resistance, Vyas produced homozygous double mutantsthat no longer displayed the hyper-resistance to fluconazole orcycloheximide displayed by the parental clinical isolate Can90. Vyasalso obtained homozygous loss-of-function mutations in essential genesof C. albicans by creating conditional alleles. Null alleles of DCR1,which is required for ribosomal RNA processing, are lethal at lowtemperature but viable at high temperature. Vyas used a repair templatethat introduced a nonsense mutation and isolated dcr1/dcr1 mutants thatfailed to grow at 16° C.

Treating Diseases with Genetic or Epigenetic Aspects

The CRISPR-Cas systems of the present invention can be used to correctgenetic mutations that were previously attempted with limited successusing TALEN and ZFN and have been identified as potential targets forCas9 systems, including as in published applications of Editas Medicinedescribing methods to use Cas9 systems to target loci to therapeuticallyaddress diseases with gene therapy, including, WO 2015/048577CRISPR-RELATED METHODS AND COMPOSITIONS of Gluckmann et al.; WO2015/070083 CRISPR-RELATED METHODS AND COMPOSITIONS WITH GOVERNING gRNASof Glucksmann et al.; In some embodiments, the treatment, prophylaxis ordiagnosis of Primary Open Angle Glaucoma (POAG) is provided. The targetis preferably the MYOC gene. This is described in WO2015153780, thedisclosure of which is hereby incorporated by reference.

Mention is made of WO2015/134812 CRISPR/CAS-RELATED METHODS ANDCOMPOSITIONS FOR TREATING USHER SYNDROME AND RETINITIS PIGMENTOSA ofMaeder et al. Through the teachings herein the invention comprehendsmethods and materials of these documents applied in conjunction with theteachings herein. In an aspect of ocular and auditory gene therapy,methods and compositions for treating Usher Syndrome andRetinitis-Pigmentosa may be adapted to the CRISPR-Cas system of thepresent invention (see, e.g., WO 2015/134812). In an embodiment, the WO2015/134812 involves a treatment or delaying the onset or progression ofUsher Syndrome type HA (USH2A, USHl1A) and retinitis pigmentosa 39(RP39) by gene editing, e.g., using CRISPR-Cas9 mediated methods tocorrect the guanine deletion at position 2299 in the USH2A gene (e.g.,replace the deleted guanine residue at position 2299 in the USH2Agene).A similar effect can be achieved with C2c1 or C2c3. In a relatedaspect, a mutation is targeted by cleaving with either one or morenuclease, one or more nickase, or a combination thereof, e.g., to induceHDR with a donor template that corrects the point mutation (e.g., thesingle nucleotide, e.g., guanine, deletion). The alteration orcorrection of the mutant USH2A gene can be mediated by any mechanism.Exemplary mechanisms that can be associated with the alteration (e.g.,correction) of the mutant HSH2A gene include, but are not limited to,non-homologous end joining, microhomology-mediated end joining (MMEJ),homology-directed repair (e.g., endogenous donor template mediated),SDSA (synthesis dependent strand annealing), single-strand annealing orsingle strand invasion. In an embodiment, the method used for treatingUsher Syndrome and Retinitis-Pigmentosa can include acquiring knowledgeof the mutation carried by the subject, e.g., by sequencing theappropriate portion of the USH2A gene.

Mention is also made of WO 2015/138510 and through the teachings hereinthe invention (using a CRISPR-Cas9 system) comprehends providing atreatment or delaying the onset or progression of Leber's CongenitalAmaurosis 10 (LCA 10). LCA 10 is caused by a mutation in the CEP290gene, e.g., a c.2991+1655, adenine to guanine mutation in the CEP290gene which gives rise to a cryptic splice site in intron 26. This is amutation at nucleotide 1655 of intron 26 of CEP290, e.g., an A to Gmutation. CEP290 is also known as: CT87; MKS4; POC3; rd16; BBS14; JBTS5;LCAJO; NPHP6; SLSN6; and 3H11Ag (see, e.g., WO 2015/138510). In anaspect of gene therapy, the invention involves introducing one or morebreaks near the site of the LCA target position (e.g., c.2991+1655; A toG) in at least one allele of the CEP290 gene. Altering the LCA10 targetposition refers to (1) break-induced introduction of an indel (alsoreferred to herein as NHEJ-mediated introduction of an indel) in closeproximity to or including a LCA10 target position (e.g., c.2991+1655A toG), or (2) break-induced deletion (also referred to herein asNHEJ-mediated deletion) of genomic sequence including the mutation at aLCA10 target position (e.g., c.2991+1655A to G). Both approaches giverise to the loss or destruction of the cryptic splice site resultingfrom the mutation at the LCA 10 target position.

Researchers are contemplating whether gene therapies could be employedto treat a wide range of diseases. The CRISPR systems of the presentinvention based on C2c1 or C2c3 effector protein are envisioned for suchtherapeutic uses, including, but noted limited to further exemplifiedtargeted areas and with delivery methods as below. Some examples ofconditions or diseases that might be usefully treated using the presentsystem are included in the examples of genes and references includedherein and are currently associated with those conditions are alsoprovided there. The genes and conditions exemplified are not exhaustive.

Treating Diseases of the Circulatory System

The present invention also contemplates delivering the CRISPR-Cassystem, specifically the novel CRISPR effector protein systems describedherein, to the blood or hematopoietic stem cells. The plasma exosomes ofWahlgren et al. (Nucleic Acids Research, 2012, Vol. 40, No. 17 e130)were previously described and may be utilized to deliver the CRISPR Cassystem to the blood. The nucleic acid-targeting system of the presentinvention is also contemplated to treat hemoglobinopathies, such asthalassemias and sickle cell disease. See, e.g., International PatentPublication No. WO 2013/126794 for potential targets that may betargeted by the CRISPR Cas system of the present invention.

Drakopoulou, “Review Article, The Ongoing Challenge of HematopoieticStem Cell-Based Gene Therapy for β-Thalassemia,” Stem CellsInternational, Volume 2011, Article ID 987980, 10 pages,doi:10.4061/2011/987980, incorporated herein by reference along with thedocuments it cites, as if set out in full, discusses modifying HSCsusing a lentivirus that delivers a gene for β-globin or γ-globin. Incontrast to using lentivirus, with the knowledge in the art and theteachings in this disclosure, the skilled person can correct HSCs as toβ-Thalassemia using a CRISPR-Cas system that targets and corrects themutation (e.g., with a suitable HDR template that delivers a codingsequence for β-globin or γ-globin, advantageously non-sickling β-globinor γ-globin); specifically, the guide RNA can target mutation that giverise to β-Thalassemia, and the HDR can provide coding for properexpression of β-globin or γ-globin. An guide RNA that targets themutation-and-Cas protein containing particle is contacted with HSCscarrying the mutation. The particle also can contain a suitable HDRtemplate to correct the mutation for proper expression of β-globin orγ-globin; or the HSC can be contacted with a second particle or a vectorthat contains or delivers the HDR template. The so contacted cells canbe administered; and optionally treated/expanded; cf. Cartier. In thisregard mention is made of: Cavazzana, “Outcomes of Gene Therapy forβ-Thalassemia Major via Transplantation of Autologous Hematopoietic StemCells Transduced Ex Vivo with a Lentiviral β^(A-T87Q)-Globin Vector.”tif2014.org/abstractFiles/Jean%20Antoine %20Ribeil_Abstract.pdf;Cavazzana-Calvo, “Transfusion independence and HMGA2 activation aftergene therapy of human β-thalassaemia”, Nature 467, 318-322 (16 Sep.2010) doi:10.1038/nature09328; Nienhuis, “Development of Gene Therapyfor Thalassemia, Cold Spring Harbor Perspectives in Medicine, doi:10.1101/cshperspect.a011833 (2012), LentiGlobin BB305, a lentiviralvector containing an engineered β-globin gene (βA-T87Q); and Xie et al.,“Seamless gene correction of β-thalassaemia mutations inpatient-specific iPSCs using CRISPR/Cas9 and piggyback” Genome Researchgr.173427.114 (2014) www.genome.org/cgi/doi/10.1101,/gr.173427.114 (ColdSpring Harbor Laboratory Press); that is the subject of Cavazzana workinvolving human β-thalassaemia and the subject of the Xie work, are allincorporated herein by reference, together with all documents citedtherein or associated therewith. In the instant invention, the HDRtemplate can provide for the HSC to express an engineered β-globin gene(e.g., βA-T87Q), or β-globin as in Xie.

Xu et al. (Sci Rep. 2015 Jul. 9; 5:12065. doi: 10.1038/srep12065) havedesigned TALENs and CRISPR-Cas9 to directly target the intron2 mutationsite IVS2-654 in the globin gene. Xu et al. observed differentfrequencies of double-strand breaks (DSBs) at IVS2-654 loci using TALENsand CRISPR-Cas9, and TALENs mediated a higher homologous gene targetingefficiency compared to CRISPR-Cas9 when combined with the piggyBactransposon donor. In addition, more obvious off-target events wereobserved for CRISPR-Cas9 compared to TALENs. Finally, TALENs-correctediPSC clones were selected for erythroblast differentiation using the OP9co-culture system and detected relatively higher transcription of HBBthan the uncorrected cells.

Song et al. (Stem Cells Dev. 2015 May 1; 24(9):1053-65. doi:10.1089/scd.2014.0347. Epub 2015 Feb. 5) used CRISPR/Cas9 to correctβ-Thal iPSCs; gene-corrected cells exhibit normal karyotypes and fullpluripotency as human embryonic stem cells (hESCs) showed nooff-targeting effects. Then, Song et al. evaluated the differentiationefficiency of the gene-corrected β-Thal iPSCs. Song et al. found thatduring hematopoietic differentiation, gene-corrected β-Thal iPSCs showedan increased embryoid body ratio and various hematopoietic progenitorcell percentages. More importantly, the gene-corrected β-Thal iPSC linesrestored HBB expression and reduced reactive oxygen species productioncompared with the uncorrected group. Song et al.'s study suggested thathematopoietic differentiation efficiency of β-Thal iPSCs was greatlyimproved once corrected by the CRISPR-Cas9 system. Similar methods maybe performed utilizing the CRISPR-Cas systems described herein, e.g.systems comprising C2c1 or C2c3 effector proteins.

Sickle cell anemia is an autosomal recessive genetic disease in whichred blood cells become sickle-shaped. It is caused by a single basesubstitution in the β-globin gene, which is located on the short arm ofchromosome 11. As a result, valine is produced instead of glutamic acidcausing the production of sickle hemoglobin (HbS). This results in theformation of a distorted shape of the erythrocytes. Due to this abnormalshape, small blood vessels can be blocked, causing serious damage to thebone, spleen and skin tissues. This may lead to episodes of pain,frequent infections, hand-foot syndrome or even multiple organ failure.The distorted erythrocytes are also more susceptible to hemolysis, whichleads to serious anemia. As in the case of β-thalassaemia, sickle cellanemia can be corrected by modifying HSCs with the CRISPR-Cas system.The system allows the specific editing of the cell's genome by cuttingits DNA and then letting it repair itself. The Cas protein is insertedand directed by a RNA guide to the mutated point and then it cuts theDNA at that point. Simultaneously, a healthy version of the sequence isinserted. This sequence is used by the cell's own repair system to fixthe induced cut. In this way, the CRISPR-Cas allows the correction ofthe mutation in the previously obtained stem cells. With the knowledgein the art and the teachings in this disclosure, the skilled person cancorrect HSCs as to sickle cell anemia using a CRISPR-Cas system thattargets and corrects the mutation (e.g., with a suitable HDR templatethat delivers a coding sequence for β-globin, advantageouslynon-sickling β-globin); specifically, the guide RNA can target mutationthat give rise to sickle cell anemia, and the HDR can provide coding forproper expression of β-globin. An guide RNA that targets themutation-and-Cas protein containing particle is contacted with HSCscarrying the mutation. The particle also can contain a suitable HDRtemplate to correct the mutation for proper expression of β-globin; orthe HSC can be contacted with a second particle or a vector thatcontains or delivers the HDR template. The so contacted cells can beadministered; and optionally treated/expanded; cf. Cartier. The HDRtemplate can provide for the HSC to express an engineered β-globin gene(e.g., PA-T87Q), or β-globin as in Xie.

Williams, “Broadening the Indications for Hematopoietic Stem CellGenetic Therapies,” Cell Stem Cell 13:263-264 (2013), incorporatedherein by reference along with the documents it cites, as if set out infull, report lentivirus-mediated gene transfer into HSC/P cells frompatients with the lysosomal storage disease metachromatic leukodystrophydisease (MLD), a genetic disease caused by deficiency of arylsulfatase A(ARSA), resulting in nerve demyelination; and lentivirus-mediated genetransfer into HSCs of patients with Wiskott-Aldrich syndrome (WAS)(patients with defective WAS protein, an effector of the small GTPaseCDC42 that regulates cytoskeletal function in blood cell lineages andthus suffer from immune deficiency with recurrent infections, autoimmunesymptoms, and thrombocytopenia with abnormally small and dysfunctionalplatelets leading to excessive bleeding and an increased risk ofleukemia and lymphoma). In contrast to using lentivirus, with theknowledge in the art and the teachings in this disclosure, the skilledperson can correct HSCs as to MLD (deficiency of arylsulfatase A (ARSA))using a CRISPR-Cas system that targets and corrects the mutation(deficiency of arylsulfatase A (ARSA)) (e.g., with a suitable HDRtemplate that delivers a coding sequence for ARSA); specifically, theguide RNA can target mutation that gives rise to MLD (deficient ARSA),and the HDR can provide coding for proper expression of ARSA. An guideRNA that targets the mutation-and-Cas protein containing particle iscontacted with HSCs carrying the mutation. The particle also can containa suitable HDR template to correct the mutation for proper expression ofARSA; or the HSC can be contacted with a second particle or a vectorthat contains or delivers the HDR template. The so contacted cells canbe administered; and optionally treated/expanded; cf. Cartier. Incontrast to using lentivirus, with the knowledge in the art and theteachings in this disclosure, the skilled person can correct HSCs as toWAS using a CRISPR-Cas system that targets and corrects the mutation(deficiency of WAS protein) (e.g., with a suitable HDR template thatdelivers a coding sequence for WAS protein); specifically, the guide RNAcan target mutation that gives rise to WAS (deficient WAS protein), andthe HDR can provide coding for proper expression of WAS protein. Anguide RNA that targets the mutation-and-C2c1 or the mutation-and-C2c3protein containing particle is contacted with HSCs carrying themutation. The particle also can contain a suitable HDR template tocorrect the mutation for proper expression of WAS protein; or the HSCcan be contacted with a second particle or a vector that contains ordelivers the HDR template. The so contacted cells can be administered;and optionally treated/expanded; cf. Cartier.

Watts, “Hematopoietic Stem Cell Expansion and Gene Therapy” Cytotherapy13(10):1164-1171. doi:10.3109/14653249.2011.620748 (2011), incorporatedherein by reference along with the documents it cites, as if set out infull, discusses hematopoietic stem cell (HSC) gene therapy, e.g.,virus-mediated HSC gene therapy, as an highly attractive treatmentoption for many disorders including hematologic conditions,immunodeficiencies including HIV/AIDS, and other genetic disorders likelysosomal storage diseases, including SCID-X1, ADA-SCID, β-thalassemia,X-linked CGD, Wiskott-Aldrich syndrome, Fanconi anemia,adrenoleukodystrophy (ALD), and metachromatic leukodystrophy (MLD).

US Patent Publication Nos. 20110225664, 20110091441, 20100229252,20090271881 and 20090222937 assigned to Cellectis, relates to CREIvariants, wherein at least one of the two I-CreI monomers has at leasttwo substitutions, one in each of the two functional subdomains of theLAGLIDADG (SEQ ID NO: 36) core domain situated respectively frompositions 26 to 40 and 44 to 77 of I-CreI, said variant being able tocleave a DNA target sequence from the human interleukin-2 receptor gammachain (IL2RG) gene also named common cytokine receptor gamma chain geneor gamma C gene. The target sequences identified in US PatentPublication Nos. 20110225664, 20110091441, 20100229252, 20090271881 and20090222937 may be utilized for the nucleic acid-targeting system of thepresent invention.

Severe Combined Immune Deficiency (SCID) results from a defect inlymphocytes T maturation, always associated with a functional defect inlymphocytes B (Cavazzana-Calvo et al., Annu. Rev. Med., 2005, 56,585-602; Fischer et al., Immunol. Rev., 2005, 203, 98-109). Overallincidence is estimated to 1 in 75 000 births. Patients with untreatedSCID are subject to multiple opportunist micro-organism infections, anddo generally not live beyond one year. SCID can be treated by allogenichematopoietic stem cell transfer, from a familial donor.Histocompatibility with the donor can vary widely. In the case ofAdenosine Deaminase (ADA) deficiency, one of the SCID forms, patientscan be treated by injection of recombinant Adenosine Deaminase enzyme.

Since the ADA gene has been shown to be mutated in SCID patients(Giblett et al., Lancet, 1972, 2, 1067-1069), several other genesinvolved in SCID have been identified (Cavazzana-Calvo et al., Annu.Rev. Med., 2005, 56, 585-602; Fischer et al., Immunol. Rev., 2005, 203,98-109). There are four major causes for SCID: (i) the most frequentform of SCID, SCID-X1 (X-linked SCID or X-SCID), is caused by mutationin the IL2RG gene, resulting in the absence of mature T lymphocytes andNK cells. IL2RG encodes the gamma C protein (Noguchi, et al., Cell,1993, 73, 147-157), a common component of at least five interleukinreceptor complexes. These receptors activate several targets through theJAK3 kinase (Macchi et al., Nature, 1995, 377, 65-68), whichinactivation results in the same syndrome as gamma C inactivation; (ii)mutation in the ADA gene results in a defect in purine metabolism thatis lethal for lymphocyte precursors, which in turn results in the quasiabsence of B, T and NK cells; (iii) V(D)J recombination is an essentialstep in the maturation of immunoglobulins and T lymphocytes receptors(TCRs). Mutations in Recombination Activating Gene 1 and 2 (RAG1 andRAG2) and Artemis, three genes involved in this process, result in theabsence of mature T and B lymphocytes; and (iv) Mutations in other genessuch as CD45, involved in T cell specific signaling have also beenreported, although they represent a minority of cases (Cavazzana-Calvoet al., Annu. Rev. Med., 2005, 56, 585-602; Fischer et al., Immunol.Rev., 2005, 203, 98-109). Since when their genetic bases have beenidentified, the different SCID forms have become a paradigm for genetherapy approaches (Fischer et al., Immunol. Rev., 2005, 203, 98-109)for two major reasons. First, as in all blood diseases, an ex vivotreatment can be envisioned. Hematopoietic Stem Cells (HSCs) can berecovered from bone marrow, and keep their pluripotent properties for afew cell divisions. Therefore, they can be treated in vitro, and thenreinjected into the patient, where they repopulate the bone marrow.Second, since the maturation of lymphocytes is impaired in SCIDpatients, corrected cells have a selective advantage. Therefore, a smallnumber of corrected cells can restore a functional immune system. Thishypothesis was validated several times by (i) the partial restoration ofimmune functions associated with the reversion of mutations in SCIDpatients (Hirschhorn et al., Nat. Genet., 1996, 13, 290-295; Stephan etal., N. Engl. J. Med., 1996, 335, 1563-1567; Bousso et al., Proc. Natl.,Acad. Sci. USA, 2000, 97, 274-278; Wada et al., Proc. Natl. Acad. Sci.USA, 2001, 98, 8697-8702; Nishikomori et al., Blood, 2004, 103,4565-4572), (ii) the correction of SCID-X1 deficiencies in vitro inhematopoietic cells (Candotti et al., Blood 1996, 87, 3097-3102;Cavazzana-Calvo et al., Blood, 1996, Blood, 88, 3901-3909; Taylor etal., Blood, 1996, 87, 3103-3107; Hacein-Bey et al., Blood, 1998, 92,4090-4097), (iii) the correction of SCID-X1 (Soudais et al., Blood,2000, 95, 3071-3077; Tsai et al., Blood, 2002, 100, 72-79), JAK-3(Bunting et al., Nat. Med., 1998, 4, 58-64; Bunting et al., Hum. GeneTher., 2000, 11, 2353-2364) and RAG2 (Yates et al., Blood, 2002, 100,3942-3949) deficiencies in vivo in animal models and (iv) by the resultof gene therapy clinical trials (Cavazzana-Calvo et al., Science, 2000,288, 669-672; Aiuti et al., Nat. Med., 2002; 8, 423-425; Gaspar et al.,Lancet, 2004, 364, 2181-2187).

US Patent Publication No. 20110182867 assigned to the Children's MedicalCenter Corporation and the President and Fellows of Harvard Collegerelates to methods and uses of modulating fetal hemoglobin expression(HbF) in a hematopoietic progenitor cells via inhibitors of BCL11Aexpression or activity, such as RNAi and antibodies. The targetsdisclosed in US Patent Publication No. 20110182867, such as BCL11A, maybe targeted by the CRISPR Cas system of the present invention formodulating fetal hemoglobin expression. See also Bauer et al. (Science11 Oct. 2013: Vol. 342 no. 6155 pp. 253-257) and Xu et al. (Science 18Nov. 2011: Vol. 334 no. 6058 pp. 993-996) for additional BCL11A targets.

With the knowledge in the art and the teachings in this disclosure, theskilled person can correct HSCs as to a genetic hematologic disorder,e.g., β-Thalassemia, Hemophilia, or a genetic lysosomal storage disease.

HSC-Delivery to and Editing of Hematopoietic Stem Cells; and ParticularConditions

The term “Hematopoietic Stem Cell” or “HSC” is meant to include broadlythose cells considered to be an HSC, e.g., blood cells that give rise toall the other blood cells and are derived from mesoderm; located in thered bone marrow, which is contained in the core of most bones. HSCs ofthe invention include cells having a phenotype of hematopoietic stemcells, identified by small size, lack of lineage (lin) markers, andmarkers that belong to the cluster of differentiation series, like:CD34, CD38, CD90, CD133, CD105, CD45, and also c-kit,—the receptor forstem cell factor. Hematopoietic stem cells are negative for the markersthat are used for detection of lineage commitment, and are, thus, calledLin-; and, during their purification by FACS, a number of up to 14different mature blood-lineage markers, e.g., CD13 & CD33 for myeloid,CD71 for erythroid, CD19 for B cells, CD61 for megakaryocytic, etc. forhumans; and, B220 (murine CD45) for B cells, Mac-1 (CD11b/CD18) formonocytes, Gr-1 for Granulocytes, Ter119 for erythroid cells, Il7Ra,CD3, CD4, CD5, CD8 for T cells, etc. Mouse HSC markers: CD34lo/−,SCA-1+, Thy1.1+/lo, CD38+, C-kit+, lin−, and Human HSC markers: CD34+,CD59+, Thy1/CD90+, CD38lo/−, C-kit/CD117+, and lin−. HSCs are identifiedby markers. Hence in embodiments discussed herein, the HSCs can be CD34+cells. HSCs can also be hematopoietic stem cells that are CD34−/CD38−.Stem cells that may lack c-kit on the cell surface that are consideredin the art as HSCs are within the ambit of the invention, as well asCD133+ cells likewise considered HSCs in the art.

The CRISPR-Cas (e.g., C2c1 or C2c3) system may be engineered to targetgenetic locus or loci in HSCs. Cas (e.g., C2c1 or C2c3) protein,advantageously codon-optimized for a eukaryotic cell and especially amammalian cell, e.g., a human cell, for instance, HSC, and sgRNAtargeting a locus or loci in HSC, e.g., the gene EMX1, may be prepared.These may be delivered via particles. The particles may be formed by theCas (e.g., C2c1 or C2c3) protein and the gRNA being admixed. The gRNAand Cas (e.g., C2c1 or C2c3) protein mixture may for example be admixedwith a mixture comprising or consisting essentially of or consisting ofsurfactant, phospholipid, biodegradable polymer, lipoprotein andalcohol, whereby particles containing the gRNA and Cas (e.g., C2c1 orC2c3) protein may be formed. The invention comprehends so makingparticles and particles from such a method as well as uses thereof.

More generally, particles may be formed using an efficient process.First, Cas (e.g., C2c1 or C2c3) protein and gRNA targeting the gene EMX1or the control gene LacZ may be mixed together at a suitable, e.g., 3:1to 1:3 or 2:1 to 1:2 or 1:1 molar ratio, at a suitable temperature,e.g., 15-30C, e.g., 20-25C, e.g., room temperature, for a suitable time,e.g., 15-45, such as 30 minutes, advantageously in sterile, nucleasefree buffer, e.g., 1×PBS. Separately, particle components such as orcomprising: a surfactant, e.g., cationic lipid, e.g.,1,2-dioleoyl-3-trimethylammonium-propane (DOTAP); phospholipid, e.g.,dimyristoylphosphatidylcholine (DMPC); biodegradable polymer, such as anethylene-glycol polymer or PEG, and a lipoprotein, such as a low-densitylipoprotein, e.g., cholesterol may be dissolved in an alcohol,advantageously a C1-6 alkyl alcohol, such as methanol, ethanol,isopropanol, e.g., 100% ethanol. The two solutions may be mixed togetherto form particles containing the Cas (e.g., C2c1 or C2c3)-gRNAcomplexes. In certain embodiments the particle can contain an HDRtemplate. That can be a particle co-administered with gRNA+Cas (e.g.,C2c1 or C2c3) protein-containing particle, or i.e., in addition tocontacting an HSC with an gRNA+Cas (e.g., C2c1 or C2c3)protein-containing particle, the HSC is contacted with a particlecontaining an HDR template; or the HSC is contacted with a particlecontaining all of the gRNA, Cas (e.g., C2c1 or C2c3) and the HDRtemplate. The HDR template can be administered by a separate vector,whereby in a first instance the particle penetrates an HSC cell and theseparate vector also penetrates the cell, wherein the HSC genome ismodified by the gRNA+Cas (e.g., C2c1 or C2c3) and the HDR template isalso present, whereby a genomic loci is modified by the HDR; forinstance, this may result in correcting a mutation.

After the particles form, HSCs in 96 well plates may be transfected with15 ug Cas (e.g., C2c1 or C2c3) protein per well. Three days aftertransfection, HSCs may be harvested, and the number of insertions anddeletions (indels) at the EMX1 locus may be quantified.

This illustrates how HSCs can be modified using CRISPR-Cas (e.g., C2c1or C2c3) targeting a genomic locus or loci of interest in the HSC. TheHSCs that are to be modified can be in vivo, i.e., in an organism, forexample a human or a non-human eukaryote, e.g., animal, such as fish,e.g., zebra fish, mammal, e.g., primate, e.g., ape, chimpanzee, macaque,rodent, e.g., mouse, rabbit, rat, canine or dog, livestock (cow/bovine,sheep/ovine, goat or pig), fowl or poultry, e.g., chicken. The HSCs thatare to be modified can be in vitro, i.e., outside of such an organism.And, modified HSCs can be used ex vivo, i.e., one or more HSCs of suchan organism can be obtained or isolated from the organism, optionallythe HSC(s) can be expanded, the HSC(s) are modified by a compositioncomprising a CRISPR-Cas (e.g., C2c1 or C2c3) that targets a geneticlocus or loci in the HSC, e.g., by contacting the HSC(s) with thecomposition, for instance, wherein the composition comprises a particlecontaining the CRISPR enzyme and one or more gRNA that targets thegenetic locus or loci in the HSC, such as a particle obtained orobtainable from admixing an gRNA and Cas (e.g., C2c1 or C2C3) proteinmixture with a mixture comprising or consisting essentially of orconsisting of surfactant, phospholipid, biodegradable polymer,lipoprotein and alcohol (wherein one or more gRNA targets the geneticlocus or loci in the HSC), optionally expanding the resultant modifiedHSCs and administering to the organism the resultant modified HSCs. Insome instances the isolated or obtained HSCs can be from a firstorganism, such as an organism from a same species as a second organism,and the second organism can be the organism to which the resultantmodified HSCs are administered, e.g., the first organism can be a donor(such as a relative as in a parent or sibling) to the second organism.Modified HSCs can have genetic modifications to address or alleviate orreduce symptoms of a disease or condition state of an individual orsubject or patient. Modified HSCs, e.g., in the instance of a firstorganism donor to a second organism, can have genetic modifications tohave the HSCs have one or more proteins e.g. surface markers or proteinsmore like that of the second organism. Modified HSCs can have geneticmodifications to simulate a disease or condition state of an individualor subject or patient and would be re-administered to a non-humanorganism so as to prepare an animal model. Expansion of HSCs is withinthe ambit of the skilled person from this disclosure and knowledge inthe art, see e.g., Lee, “Improved ex vivo expansion of adulthematopoietic stem cells by overcoming CUL4-mediated degradation ofHOXB4.” Blood. 2013 May 16; 121(20):4082-9. doi:10.1182/blood-2012-09-455204. Epub 2013 Mar. 21.

As indicated to improve activity, gRNA may be pre-complexed with the Cas(e.g., C2c1 or C2c3) protein, before formulating the entire complex in aparticle. Formulations may be made with a different molar ratio ofdifferent components known to promote delivery of nucleic acids intocells (e.g. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP),1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC), polyethyleneglycol (PEG), and cholesterol) For example DOTAP: DMPC: PEG: CholesterolMolar Ratios may be DOTAP 100, DMPC 0, PEG 0, Cholesterol 0; or DOTAP90, DMPC 0, PEG 10, Cholesterol 0; or DOTAP 90, DMPC 0, PEG 5,Cholesterol 5. DOTAP 100, DMPC 0, PEG 0, Cholesterol 0. The inventionaccordingly comprehends admixing gRNA, Cas (e.g., C2c1 or C2c3) proteinand components that form a particle; as well as particles from suchadmixing.

In a preferred embodiment, particles containing the Cas (e.g., C2c1 orC2c3)-gRNA complexes may be formed by mixing Cas (e.g., C2c1 or C2c3)protein and one or more gRNAs together, preferably at a 1:1 molar ratio,enzyme: guide RNA. Separately, the different components known to promotedelivery of nucleic acids (e.g. DOTAP, DMPC, PEG, and cholesterol) aredissolved, preferably in ethanol. The two solutions are mixed togetherto form particles containing the Cas (e.g., C2c1 or C2c3)-gRNAcomplexes. After the particles are formed, Cas (e.g., C2c1 or C2c3)-gRNAcomplexes may be transfected into cells (e.g. HSCs). Bar coding may beapplied. The particles, the Cas-9 and/or the gRNA may be barcoded.

The invention in an embodiment comprehends a method of preparing angRNA-and-Cas (e.g., C2c1 or C2c3) protein containing particle comprisingadmixing an gRNA and Cas (e.g., C2c1 or C2c3) protein mixture with amixture comprising or consisting essentially of or consisting ofsurfactant, phospholipid, biodegradable polymer, lipoprotein andalcohol. An embodiment comprehends an gRNA-and-Cas (e.g., C2c1 or C2c3)protein containing particle from the method. The invention in anembodiment comprehends use of the particle in a method of modifying agenomic locus of interest, or an organism or a non-human organism bymanipulation of a target sequence in a genomic locus of interest,comprising contacting a cell containing the genomic locus of interestwith the particle wherein the gRNA targets the genomic locus ofinterest; or a method of modifying a genomic locus of interest, or anorganism or a non-human organism by manipulation of a target sequence ina genomic locus of interest, comprising contacting a cell containing thegenomic locus of interest with the particle wherein the gRNA targets thegenomic locus of interest. In these embodiments, the genomic locus ofinterest is advantageously a genomic locus in an HSC.

Considerations for Therapeutic Applications: A consideration in genomeediting therapy is the choice of sequence-specific nuclease, such as avariant of a C2c1 or C2c3 nuclease. Each nuclease variant may possessits own unique set of strengths and weaknesses, many of which must bebalanced in the context of treatment to maximize therapeutic benefit.Thus far, two therapeutic editing approaches with nucleases have shownsignificant promise: gene disruption and gene correction. Genedisruption involves stimulation of NHEJ to create targeted indels ingenetic elements, often resulting in loss of function mutations that arebeneficial to patients. In contrast, gene correction uses HDR todirectly reverse a disease causing mutation, restoring function whilepreserving physiological regulation of the corrected element. HDR mayalso be used to insert a therapeutic transgene into a defined ‘safeharbor’ locus in the genome to recover missing gene function. For aspecific editing therapy to be efficacious, a sufficiently high level ofmodification must be achieved in target cell populations to reversedisease symptoms. This therapeutic modification ‘threshold’ isdetermined by the fitness of edited cells following treatment and theamount of gene product necessary to reverse symptoms. With regard tofitness, editing creates three potential outcomes for treated cellsrelative to their unedited counterparts: increased, neutral, ordecreased fitness. In the case of increased fitness, for example in thetreatment of SCID-X1, modified hematopoietic progenitor cellsselectively expand relative to their unedited counterparts. SCID-X1 is adisease caused by mutations in the IL2RG gene, the function of which isrequired for proper development of the hematopoietic lymphocyte lineage[Leonard, W. J., et al. Immunological reviews 138, 61-86 (1994);Kaushansky, K. & Williams, W. J. Williams hematology, (McGraw-HillMedical, New York, 2010)]. In clinical trials with patients who receivedviral gene therapy for SCID-X1, and a rare example of a spontaneouscorrection of SCID-X1 mutation, corrected hematopoietic progenitor cellsmay be able to overcome this developmental block and expand relative totheir diseased counterparts to mediate therapy [Bousso, P., et al.Proceedings of the National Academy of Sciences of the United States ofAmerica 97, 274-278 (2000); Hacein-Bey-Abina, S., et al. The New Englandjournal of medicine 346, 1185-1193 (2002); Gaspar, H. B., et al. Lancet364, 2181-2187 (2004)]. In this case, where edited cells possess aselective advantage, even low numbers of edited cells can be amplifiedthrough expansion, providing a therapeutic benefit to the patient. Incontrast, editing for other hematopoietic diseases, like chronicgranulomatous disorder (CGD), would induce no change in fitness foredited hematopoietic progenitor cells, increasing the therapeuticmodification threshold. CGD is caused by mutations in genes encodingphagocytic oxidase proteins, which are normally used by neutrophils togenerate reactive oxygen species that kill pathogens [Mukherjee, S. &Thrasher, A. J. Gene 525, 174-181 (2013)]. As dysfunction of these genesdoes not influence hematopoietic progenitor cell fitness or development,but only the ability of a mature hematopoietic cell type to fightinfections, there would be likely no preferential expansion of editedcells in this disease. Indeed, no selective advantage for gene correctedcells in CGD has been observed in gene therapy trials, leading todifficulties with long-term cell engraftment [Malech, H. L., et al.Proceedings of the National Academy of Sciences of the United States ofAmerica 94, 12133-12138 (1997); Kang, H. J., et al. Molecular therapy:the journal of the American Society of Gene Therapy 19, 2092-2101(2011)]. As such, significantly higher levels of editing would berequired to treat diseases like CGD, where editing creates a neutralfitness advantage, relative to diseases where editing creates increasedfitness for target cells. If editing imposes a fitness disadvantage, aswould be the case for restoring function to a tumor suppressor gene incancer cells, modified cells would be outcompeted by their diseasedcounterparts, causing the benefit of treatment to be low relative toediting rates. This latter class of diseases would be particularlydifficult to treat with genome editing therapy.

In addition to cell fitness, the amount of gene product necessary totreat disease also influences the minimal level of therapeutic genomeediting that must be achieved to reverse symptoms. Haemophilia B is onedisease where a small change in gene product levels can result insignificant changes in clinical outcomes. This disease is caused bymutations in the gene encoding factor IX, a protein normally secreted bythe liver into the blood, where it functions as a component of theclotting cascade. Clinical severity of haemophilia B is related to theamount of factor IX activity. Whereas severe disease is associated withless than 1% of normal activity, milder forms of the diseases areassociated with greater than 1% of factor IX activity [Kaushansky, K. &Williams, W. J. Williams hematology, (McGraw-Hill Medical, New York,2010); Lofqvist, T., et al. Journal Of Internal Medicine 241, 395-400(1997)]. This suggests that editing therapies that can restore factor IXexpression to even a small percentage of liver cells could have a largeimpact on clinical outcomes. A study using ZFNs to correct a mouse modelof haemophilia B shortly after birth demonstrated that 3-7% correctionwas sufficient to reverse disease symptoms, providing preclinicalevidence for this hypothesis [Li, H., et al. Nature 475, 217-221(2011)].

Disorders where a small change in gene product levels can influenceclinical outcomes and diseases where there is a fitness advantage foredited cells, are ideal targets for genome editing therapy, as thetherapeutic modification threshold is low enough to permit a high chanceof success given the current technology. Targeting these diseases hasnow resulted in successes with editing therapy at the preclinical leveland a phase I clinical trial. Improvements in DSB repair pathwaymanipulation and nuclease delivery are needed to extend these promisingresults to diseases with a neutral fitness advantage for edited cells,or where larger amounts of gene product are needed for treatment. TheTable below shows some examples of applications of genome editing totherapeutic models, and the references of the below Table and thedocuments cited in those references are hereby incorporated herein byreference as if set out in full.

TABLE 8 Nuclease Platform Therapeutic Disease Type Employed StrategyReferences Hemophilia B ZFN HDR-mediated Li, H., et al. insertion ofcorrect Nature 475, gene sequence 217-221 (2011) SCID ZFN HDR-mediatedGenovese, P., et al. insertion of correct Nature 510, gene sequence235-240 (2014) Hereditary CRISPR HDR-mediated Yin, H., et al.tyrosinemia correction of mutation Nature in liver biotechnology 32,551-553 (2014)

Addressing each of the conditions of the foregoing table, using theCRISPR-Cas (e.g., C2c1 or C2c3) system to target by either HDR-mediatedcorrection of mutation, or HDR-mediated insertion of correct genesequence, advantageously via a delivery system as herein, e.g., aparticle delivery system, is within the ambit of the skilled person fromthis disclosure and the knowledge in the art. Thus, an embodimentcomprehends contacting a Hemophilia B, SCID (e.g., SCID-X1, ADA-SCID) orHereditary tyrosinemia mutation-carrying HSC with an gRNA-and-Cas (e.g.,C2c1 or C2c3) protein containing particle targeting a genomic locus ofinterest as to Hemophilia B, SCID (e.g., SCID-X1, ADA-SCID) orHereditary tyrosinemia (e.g., as in Li, Genovese or Yin). The particlealso can contain a suitable HDR template to correct the mutation; or theHSC can be contacted with a second particle or a vector that contains ordelivers the HDR template. In this regard, it is mentioned thatHaemophilia B is an X-linked recessive disorder caused byloss-of-function mutations in the gene encoding Factor IX, a crucialcomponent of the clotting cascade. Recovering Factor IX activity toabove 1% of its levels in severely affected individuals can transformthe disease into a significantly milder form, as infusion of recombinantFactor IX into such patients prophylactically from a young age toachieve such levels largely ameliorates clinical complications. With theknowledge in the art and the teachings in this disclosure, the skilledperson can correct HSCs as to Haemophilia B using a CRISPR-Cas (eg C2c1or C2c3) system that targets and corrects the mutation (X-linkedrecessive disorder caused by loss-of-function mutations in the geneencoding Factor IX) (e.g., with a suitable HDR template that delivers acoding sequence for Factor IX); specifically, the gRNA can targetmutation that give rise to Haemophilia B, and the HDR can provide codingfor proper expression of Factor IX. An gRNA that targets themutation-and-Cas (eg C2c1 or C2c3) protein containing particle iscontacted with HSCs carrying the mutation. The particle also can containa suitable HDR template to correct the mutation for proper expression ofFactor IX; or the HSC can be contacted with a second particle or avector that contains or delivers the HDR template. The so contactedcells can be administered; and optionally treated/expanded; cf. Cartier,discussed herein.

In Cartier, “MINI-SYMPOSIUM: X-Linked Adrenoleukodystrophy,Hematopoietic Stem Cell Transplantation and Hematopoietic Stem Cell GeneTherapy in X-Linked Adrenoleukodystrophy,” Brain Pathology 20 (2010)857-862, incorporated herein by reference along with the documents itcites, as if set out in full, there is recognition that allogeneichematopoietic stem cell transplantation (HSCT) was utilized to delivernormal lysosomal enzyme to the brain of a patient with Hurler's disease,and a discussion of HSC gene therapy to treat ALD. In two patients,peripheral CD34+ cells were collected after granulocyte-colonystimulating factor (G-CSF) mobilization and transduced with anmyeloproliferative sarcoma virus enhancer, negative control regiondeleted, dl587rev primer binding site substituted (MND)-ALD lentiviralvector. CD34+ cells from the patients were transduced with the MND-ALDvector during 16 h in the presence of cytokines at low concentrations.Transduced CD34+ cells were frozen after transduction to perform on 5%of cells various safety tests that included in particular threereplication-competent lentivirus (RCL) assays. Transduction efficacy ofCD34+ cells ranged from 35% to 50% with a mean number of lentiviralintegrated copy between 0.65 and 0.70. After the thawing of transducedCD34+ cells, the patients were reinfused with more than 4.106 transducedCD34+ cells/kg following full myeloablation with busulfan andcyclophos-phamide. The patient's HSCs were ablated to favor engraftmentof the gene-corrected HSCs. Hematological recovery occurred between days13 and 15 for the two patients. Nearly complete immunological recoveryoccurred at 12 months for the first patient, and at 9 months for thesecond patient. In contrast to using lentivirus, with the knowledge inthe art and the teachings in this disclosure, the skilled person cancorrect HSCs as to ALD using a CRISPR-Cas (C2c1 or C2c3) system thattargets and corrects the mutation (e.g., with a suitable HDR template);specifically, the gRNA can target mutations in ABCD1, a gene located onthe X chromosome that codes for ALD, a peroxisomal membrane transporterprotein, and the HDR can provide coding for proper expression of theprotein. An gRNA that targets the mutation-and-Cas (C2c1 or C2c3)protein containing particle is contacted with HSCs, e.g., CD34+ cellscarrying the mutation as in Cartier. The particle also can contain asuitable HDR template to correct the mutation for expression of theperoxisomal membrane transporter protein; or the HSC can be contactedwith a second particle or a vector that contains or delivers the HDRtemplate. The so contacted cells optionally can be treated as inCartier. The so contacted cells can be administered as in Cartier.

Mention is made of WO 2015/148860, through the teachings herein theinvention comprehends methods and materials of these documents appliedin conjunction with the teachings herein. In an aspect of blood-relateddisease gene therapy, methods and compositions for treating betathalassemia may be adapted to the CRISPR-Cas system of the presentinvention (see, e.g., WO 2015/148860). In an embodiment, WO 2015/148860involves the treatment or prevention of beta thalassemia, or itssymptoms, e.g., by altering the gene for B-cell CLL/lymphoma 11A(BCL11A). The BCL11A gene is also known as B-cell CLL/lymphoma 11A,BCL11A-L, BCL11A-S, BCL11AXL, CTIP 1, HBFQTL5 and ZNF. BCL11A encodes azinc-finger protein that is involved in the regulation of globin geneexpression. By altering the BCL11A gene (e.g., one or both alleles ofthe BCL11A gene), the levels of gamma globin can be increased. Gammaglobin can replace beta globin in the hemoglobin complex and effectivelycarry oxygen to tissues, thereby ameliorating beta thalassemia diseasephenotypes.

Mention is also made of WO 2015/148863 and through the teachings hereinthe invention comprehends methods and materials of these documents whichmay be adapted to the CRISPR-Cas system of the present invention. In anaspect of treating and preventing sickle cell disease, which is aninherited hematologic disease, WO 2015/148863 comprehends altering theBCL11A gene. By altering the BCL11A gene (e.g., one or both alleles ofthe BCL11A gene), the levels of gamma globin can be increased. Gammaglobin can replace beta globin in the hemoglobin complex and effectivelycarry oxygen to tissues, thereby ameliorating sickle cell diseasephenotypes.

In an aspect of the invention, methods and compositions which involveediting a target nucleic acid sequence, or modulating expression of atarget nucleic acid sequence, and applications thereof in connectionwith cancer immunotherapy are comprehended by adapting the CRISPR-Cassystem of the present invention. Reference is made to the application ofgene therapy in WO 2015/161276 which involves methods and compositionswhich can be used to affect T-cell proliferation, survival and/orfunction by altering one or more T-cell expressed genes, e.g., one ormore of FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, TRAC and/or TRBC genes. Ina related aspect, T-cell proliferation can be affected by altering oneor more T-cell expressed genes, e.g., the CBLB and/or PTPN6 gene, FASand/or BID gene, CTLA4 and/or PDCDI and/or TRAC and/or TRBC gene.

Chimeric antigen receptor (CAR)19 T-cells exhibit anti-leukemic effectsin patient malignancies. However, leukemia patients often do not haveenough T-cells to collect, meaning that treatment must involve modifiedT cells from donors. Accordingly, there is interest in establishing abank of donor T-cells. Qasim et al. (“First Clinical Application ofTalen Engineered Universal CAR19 T Cells in B-ALL” ASH 57th AnnualMeeting and Exposition, Dec. 5-8, 2015, Abstract 2046(ash.confex.com/ash/2015/webprogram/Paper81653.html published onlineNovember 2015) discusses modifying CAR19 T cells to eliminate the riskof graft-versus-host disease through the disruption of T-cell receptorexpression and CD52 targeting. Furthermore, CD52 cells were targetedsuch that they became insensitive to Alemtuzumab, and thus allowedAlemtuzumab to prevent host-mediated rejection of human leukocyteantigen (HLA) mismatched CAR19 T-cells. Investigators used thirdgeneration self-inactivating lentiviral vector encoding a 4g7 CAR19(CD19 scFv-4-1BB-CD3ζ) linked to RQR8, then electroporated cells withtwo pairs of TALEN mRNA for multiplex targeting for both the T-cellreceptor (TCR) alpha constant chain locus and the CD52 gene locus. Cellswhich were still expressing TCR following ex vivo expansion weredepleted using CliniMacs α/β TCR depletion, yielding a T-cell product(UCART19) with <1% TCR expression, 85% of which expressed CAR19, and 64%becoming CD52 negative. The modified CAR19 T cells were administered totreat a patient's relapsed acute lymphoblastic leukemia. The teachingsprovided herein provide effective methods for providing modifiedhematopoietic stem cells and progeny thereof, including but not limitedto cells of the myeloid and lymphoid lineages of blood, including Tcells, B cells, monocytes, macrophages, neutrophils, basophils,eosinophils, erythrocytes, dendritic cells, and megakaryocytes orplatelets, and natural killer cells and their precursors andprogenitors. Such cells can be modified by knocking out, knocking in, orotherwise modulating targets, for example to remove or modulate CD52 asdescribed above, and other targets, such as, without limitation, CXCR4,and PD-1. Thus compositions, cells, and method of the invention can beused to modulate immune responses and to treat, without limitation,malignancies, viral infections, and immune disorders, in conjunctionwith modification of administration of T cells or other cells topatients.

Mention is made of WO 2015/148670 and through the teachings herein theinvention comprehends methods and materials of this document applied inconjunction with the teachings herein. In an aspect of gene therapy,methods and compositions for editing of a target sequence related to orin connection with Human Immunodeficiency Virus (HIV) and AcquiredImmunodeficiency Syndrome (AIDS) are comprehended. In a related aspect,the invention described herein comprehends prevention and treatment ofHIV infection and AIDS, by introducing one or more mutations in the genefor C-C chemokine receptor type 5 (CCR5). The CCR5 gene is also known asCKR5, CCR-5, CD195, CKR-5, CCCKR5, CMKBR5, IDDM22, and CC-CKR-5. In afurther aspect, the invention described herein comprehends provide forprevention or reduction of HIV infection and/or prevention or reductionof the ability for HIV to enter host cells, e.g., in subjects who arealready infected. Exemplary host cells for HIV include, but are notlimited to, CD4 cells, T cells, gut associated lymphatic tissue (GALT),macrophages, dendritic cells, myeloid precursor cell, and microglia.Viral entry into the host cells requires interaction of the viralglycoproteins gp41 and gp120 with both the CD4 receptor and aco-receptor, e.g., CCR5. If a co-receptor, e.g., CCR5, is not present onthe surface of the host cells, the virus cannot bind and enter the hostcells. The progress of the disease is thus impeded. By knocking out orknocking down CCR5 in the host cells, e.g., by introducing a protectivemutation (such as a CCR5 delta 32 mutation), entry of the HIV virus intothe host cells is prevented.

X-linked Chronic granulomatous disease (CGD) is a hereditary disorder ofhost defense due to absent or decreased activity of phagocyte NADPHoxidase. Using a CRISPR-Cas (C2c1 or C2c3) system that targets andcorrects the mutation (absent or decreased activity of phagocyte NADPHoxidase) (e.g., with a suitable HDR template that delivers a codingsequence for phagocyte NADPH oxidase); specifically, the gRNA can targetmutation that gives rise to CGD (deficient phagocyte NADPH oxidase), andthe HDR can provide coding for proper expression of phagocyte NADPHoxidase. An gRNA that targets the mutation-and-Cas (C2c1 or C2c3)protein containing particle is contacted with HSCs carrying themutation. The particle also can contain a suitable HDR template tocorrect the mutation for proper expression of phagocyte NADPH oxidase;or the HSC can be contacted with a second particle or a vector thatcontains or delivers the HDR template. The so contacted cells can beadministered; and optionally treated/expanded; cf. Cartier.

Fanconi anemia: Mutations in at least 15 genes (FANCA, FANCB, FANCC,FANCD1/BRCA2, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ/BACH1/BRIP1,FANCL/PHF9/POG, FANCM, FANCN/PALB2, FANCO/Rad51C, and FANCP/SLX4/BTBD12)can cause Fanconi anemia. Proteins produced from these genes areinvolved in a cell process known as the FA pathway. The FA pathway isturned on (activated) when the process of making new copies of DNA,called DNA replication, is blocked due to DNA damage. The FA pathwaysends certain proteins to the area of damage, which trigger DNA repairso DNA replication can continue. The FA pathway is particularlyresponsive to a certain type of DNA damage known as interstrandcross-links (ICLs). ICLs occur when two DNA building blocks(nucleotides) on opposite strands of DNA are abnormally attached orlinked together, which stops the process of DNA replication. ICLs can becaused by a buildup of toxic substances produced in the body or bytreatment with certain cancer therapy drugs. Eight proteins associatedwith Fanconi anemia group together to form a complex known as the FAcore complex. The FA core complex activates two proteins, called FANCD2and FANCI. The activation of these two proteins brings DNA repairproteins to the area of the ICL so the cross-link can be removed and DNAreplication can continue. the FA core complex. More in particular, theFA core complex is a nuclear multiprotein complex consisting of FANCA,FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, and FANCM, functions as an E3ubiquitin ligase and mediates the activation of the ID complex, which isa heterodimer composed of FANCD2 and FANCI. Once monoubiquitinated, itinteracts with classical tumor suppressors downstream of the FA pathwayincluding FANCD1/BRCA2, FANCN/PALB2, FANCJ/BRIP1, and FANCO/Rad51C andthereby contributes to DNA repair via homologous recombination (HR).Eighty to 90 percent of FA cases are due to mutations in one of threegenes, FANCA, FANCC, and FANCG. These genes provide instructions forproducing components of the FA core complex. Mutations in such genesassociated with the FA core complex will cause the complex to benonfunctional and disrupt the entire FA pathway. As a result, DNA damageis not repaired efficiently and ICLs build up over time. Geiselhart,“Review Article, Disrupted Signaling through the Fanconi Anemia PathwayLeads to Dysfunctional Hematopoietic Stem Cell Biology: UnderlyingMechanisms and Potential Therapeutic Strategies,” Anemia Volume 2012(2012), Article ID 265790, dx.doi.org/10.1155/2012/265790 discussed FAand an animal experiment involving intrafemoral injection of alentivirus encoding the FANCC gene resulting in correction of HSCs invivo. Using a CRISPR-Cas (C2c1 or C2c3) system that targets and one ormore of the mutations associated with FA, for instance a CRISPR-Cas(C2c1 or C2c3) system having gRNA(s) and HDR template(s) thatrespectively targets one or more of the mutations of FANCA, FANCC, orFANCG that give rise to FA and provide corrective expression of one ormore of FANCA, FANCC or FANCG; e.g., the gRNA can target a mutation asto FANCC, and the HDR can provide coding for proper expression of FANCC.An gRNA that targets the mutation(s) (e.g., one or more involved in FA,such as mutation(s) as to any one or more of FANCA, FANCC orFANCG)-and-Cas (C2c1 or C2c3) protein containing particle is contactedwith HSCs carrying the mutation(s). The particle also can contain asuitable HDR template(s) to correct the mutation for proper expressionof one or more of the proteins involved in FA, such as any one or moreof FANCA, FANCC or FANCG; or the HSC can be contacted with a secondparticle or a vector that contains or delivers the HDR template. The socontacted cells can be administered; and optionally treated/expanded;cf. Cartier.

The particle in the herein discussion (e.g., as to containing gRNA(s)and Cas (C2c1 or C2c3), optionally HDR template(s), or HDR template(s);for instance as to Hemophilia B, SCID, SCID-X1, ADA-SCID, Hereditarytyrosinemia, -thalassemia, X-linked CGD, Wiskott-Aldrich syndrome,Fanconi anemia, adrenoleukodystrophy (ALD), metachromatic leukodystrophy(MLD), HIV/AIDS, Immunodeficiency disorder, Hematologic condition, orgenetic lysosomal storage disease) is advantageously obtained orobtainable from admixing an gRNA(s) and Cas (C2c1 or C2c3) proteinmixture (optionally containing HDR template(s) or such mixture onlycontaining HDR template(s) when separate particles as to template(s) isdesired) with a mixture comprising or consisting essentially of orconsisting of surfactant, phospholipid, biodegradable polymer,lipoprotein and alcohol (wherein one or more gRNA targets the geneticlocus or loci in the HSC).

Indeed, the invention is especially suited for treating hematopoieticgenetic disorders with genome editing, and immunodeficiency disorders,such as genetic immunodeficiency disorders, especially through using theparticle technology herein-discussed. Genetic immunodeficiencies arediseases where genome editing interventions of the instant invention cansuccessful. The reasons include: Hematopoietic cells, of which immunecells are a subset, are therapeutically accessible. They can be removedfrom the body and transplanted autologously or allogenically. Further,certain genetic immunodeficiencies, e.g., severe combinedimmunodeficiency (SCID), create a proliferative disadvantage for immunecells. Correction of genetic lesions causing SCID by rare, spontaneous‘reverse’ mutations indicates that correcting even one lymphocyteprogenitor may be sufficient to recover immune function in patients . .. / . . . / . . ./Users/t_kowalski/AppData/Local/Microsoft/Windows/Temporary InternetFiles/Content.Outlook/GA8VY8LK/Treating SCID for Ellen.docx-_ENREF_1 SeeBousso, P., et al. Diversity, functionality, and stability of the T cellrepertoire derived in vivo from a single human T cell precursor.Proceedings of the National Academy of Sciences of the United States ofAmerica 97, 274-278 (2000). The selective advantage for edited cellsallows for even low levels of editing to result in a therapeutic effect.This effect of the instant invention can be seen in SCID,Wiskott-Aldrich Syndrome, and the other conditions mentioned herein,including other genetic hematopoietic disorders such as alpha- andbeta-thalassemia, where hemoglobin deficiencies negatively affect thefitness of erythroid progenitors.

The activity of NHEJ and HDR DSB repair varies significantly by celltype and cell state. NHEJ is not highly regulated by the cell cycle andis efficient across cell types, allowing for high levels of genedisruption in accessible target cell populations. In contrast, HDR actsprimarily during S/G2 phase, and is therefore restricted to cells thatare actively dividing, limiting treatments that require precise genomemodifications to mitotic cells [Ciccia, A. & Elledge, S. J. Molecularcell 40, 179-204 (2010); Chapman, J. R., et al. Molecular cell 47,497-510 (2012)].

The efficiency of correction via HDR may be controlled by the epigeneticstate or sequence of the targeted locus, or the specific repair templateconfiguration (single vs. double stranded, long vs. short homology arms)used [Hacein-Bey-Abina, S., et al. The New England journal of medicine346, 1185-1193 (2002); Gaspar, H. B., et al. Lancet 364, 2181-2187(2004); Beumer, K. J., et al. G3 (2013)]. The relative activity of NHEJand HDR machineries in target cells may also affect gene correctionefficiency, as these pathways may compete to resolve DSBs [Beumer, K.J., et al. Proceedings of the National Academy of Sciences of the UnitedStates of America 105, 19821-19826 (2008)]. HDR also imposes a deliverychallenge not seen with NHEJ strategies, as it requires the concurrentdelivery of nucleases and repair templates. In practice, theseconstraints have so far led to low levels of HDR in therapeuticallyrelevant cell types. Clinical translation has therefore largely focusedon NHEJ strategies to treat disease, although proof-of-conceptpreclinical HDR treatments have now been described for mouse models ofhaemophilia B and hereditary tyrosinemia [Li, H., et al. Nature 475,217-221 (2011); Yin, H., et al. Nature biotechnology 32, 551-553(2014)].

Any given genome editing application may comprise combinations ofproteins, small RNA molecules, and/or repair templates, making deliveryof these multiple parts substantially more challenging than smallmolecule therapeutics. Two main strategies for delivery of genomeediting tools have been developed: ex vivo and in vivo. In ex vivotreatments, diseased cells are removed from the body, edited and thentransplanted back into the patient. Er vivo editing has the advantage ofallowing the target cell population to be well defined and the specificdosage of therapeutic molecules delivered to cells to be specified. Thelatter consideration may be particularly important when off-targetmodifications are a concern, as titrating the amount of nuclease maydecrease such mutations (Hsu et al., 2013). Another advantage of ex vivoapproaches is the typically high editing rates that can be achieved, dueto the development of efficient delivery systems for proteins andnucleic acids into cells in culture for research and gene therapyapplications.

There may be drawbacks with ex vivo approaches that limit application toa small number of diseases. For instance, target cells must be capableof surviving manipulation outside the body. For many tissues, like thebrain, culturing cells outside the body is a major challenge, becausecells either fail to survive, or lose properties necessary for theirfunction in vivo. Thus, in view of this disclosure and the knowledge inthe art, ex vivo therapy as to tissues with adult stem cell populationsamenable to ex vivo culture and manipulation, such as the hematopoieticsystem, by the CRISPR-Cas (C2c1 or C2c3) system are enabled. [Bunn, H.F. & Aster, J. Pathophysiology Of Blood Disorders, (McGraw-Hill, NewYork, 2011)].

In vivo genome editing involves direct delivery of editing systems tocell types in their native tissues. In vivo editing allows diseases inwhich the affected cell population is not amenable to ex vivomanipulation to be treated. Furthermore, delivering nucleases to cellsin situ allows for the treatment of multiple tissue and cell types.These properties probably allow in vivo treatment to be applied to awider range of diseases than ex vivo therapies.

To date, in vivo editing has largely been achieved through the use ofviral vectors with defined, tissue-specific tropism. Such vectors arecurrently limited in terms of cargo carrying capacity and tropism,restricting this mode of therapy to organ systems where transductionwith clinically useful vectors is efficient, such as the liver, muscleand eye [Kotterman, M. A. & Schaffer, D. V. Nature reviews. Genetics 15,445-451 (2014); Nguyen, T. H. & Ferry, N. Gene therapy 11 Suppl 1,S76-84 (2004); Boye, S. E., et al. Molecular therapy: the journal of theAmerican Society of Gene Therapy 21, 509-519 (2013)].

A potential barrier for in vivo delivery is the immune response that maybe created in response to the large amounts of virus necessary fortreatment, but this phenomenon is not unique to genome editing and isobserved with other virus based gene therapies [Bessis, N., et al. Genetherapy 11 Suppl 1, S10-17 (2004)]. It is also possible that peptidesfrom editing nucleases themselves are presented on MHC Class I moleculesto stimulate an immune response, although there is little evidence tosupport this happening at the preclinical level. Another majordifficulty with this mode of therapy is controlling the distribution andconsequently the dosage of genome editing nucleases in vivo, leading tooff-target mutation profiles that may be difficult to predict. However,in view of this disclosure and the knowledge in the art, including theuse of virus- and particle-based therapies being used in the treatmentof cancers, in vivo modification of HSCs, for instance by delivery byeither particle or virus, is within the ambit of the skilled person.

Ex Vivo Editing Therapy: The long standing clinical expertise with thepurification, culture and transplantation of hematopoietic cells hasmade diseases affecting the blood system such as SCID, Fanconi anemia,Wiskott-Aldrich syndrome and sickle cell anemia the focus of ex vivoediting therapy. Another reason to focus on hematopoietic cells is that,thanks to previous efforts to design gene therapy for blood disorders,delivery systems of relatively high efficiency already exist. With theseadvantages, this mode of therapy can be applied to diseases where editedcells possess a fitness advantage, so that a small number of engrafted,edited cells can expand and treat disease. One such disease is HIV,where infection results in a fitness disadvantage to CD4+ T cells.

Er vivo editing therapy has been recently extended to include genecorrection strategies. The barriers to HDR ex vivo were overcome in arecent paper from Genovese and colleagues, who achieved gene correctionof a mutated IL2RG gene in hematopoietic stem cells (HSCs) obtained froma patient suffering from SCID-X1 [Genovese, P., et al. Nature 510,235-240 (2014)]. Genovese et. al. accomplished gene correction in HSCsusing a multimodal strategy. First, HSCs were transduced usingintegration-deficient lentivirus containing an HDR template encoding atherapeutic cDNA for IL2RG. Following transduction, cells wereelectroporated with mRNA encoding ZFNs targeting a mutational hotspot inIL2RG to stimulate HDR based gene correction. To increase HDR rates,culture conditions were optimized with small molecules to encourage HSCdivision. With optimized culture conditions, nucleases and HDRtemplates, gene corrected HSCs from the SCID-X1 patient were obtained inculture at therapeutically relevant rates. HSCs from unaffectedindividuals that underwent the same gene correction procedure couldsustain long-term hematopoiesis in mice, the gold standard for HSCfunction. HSCs are capable of giving rise to all hematopoietic celltypes and can be autologously transplanted, making them an extremelyvaluable cell population for all hematopoietic genetic disorders[Weissman, I. L. & Shizuru, J. A. Blood 112, 3543-3553 (2008)]. Genecorrected HSCs could, in principle, be used to treat a wide range ofgenetic blood disorders making this study an exciting breakthrough fortherapeutic genome editing.

In Vivo Editing Therapy: In vivo editing can be used advantageously fromthis disclosure and the knowledge in the art. For organ systems wheredelivery is efficient, there have already been a number of excitingpreclinical therapeutic successes. The first example of successful invivo editing therapy was demonstrated in a mouse model of haemophilia B[Li, H., et al. Nature 475, 217-221 (2011)]. As noted earlier,Haemophilia B is an X-linked recessive disorder caused byloss-of-function mutations in the gene encoding Factor IX, a crucialcomponent of the clotting cascade. Recovering Factor IX activity toabove 1% of its levels in severely affected individuals can transformthe disease into a significantly milder form, as infusion of recombinantFactor IX into such patients prophylactically from a young age toachieve such levels largely ameliorates clinical complications[Lofqvist, T., et al. Journal of internal medicine 241, 395-400 (1997)].Thus, only low levels of HDR gene correction are necessary to changeclinical outcomes for patients. In addition, Factor IX is synthesizedand secreted by the liver, an organ that can be transduced efficientlyby viral vectors encoding editing systems.

Using hepatotropic adeno-associated viral (AAV) serotypes encoding ZFNsand a corrective HDR template, up to 7% gene correction of a mutated,humanized Factor IX gene in the murine liver was achieved [Li, H., etal. Nature 475, 217-221 (2011)]. This resulted in improvement of clotformation kinetics, a measure of the function of the clotting cascade,demonstrating for the first time that in vivo editing therapy is notonly feasible, but also efficacious. As discussed herein, the skilledperson is positioned from the teachings herein and the knowledge in theart, e.g., Li to address Haemophilia B with a particle-containing HDRtemplate and a CRISPR-Cas (C2c1 or C2c3) system that targets themutation of the X-linked recessive disorder to reverse theloss-of-function mutation.

Building on this study, other groups have recently used in vivo genomeediting of the liver with CRISPR-Cas to successfully treat a mouse modelof hereditary tyrosinemia and to create mutations that provideprotection against cardiovascular disease. These two distinctapplications demonstrate the versatility of this approach for disordersthat involve hepatic dysfunction [Yin, H., et al. Nature biotechnology32, 551-553 (2014); Ding, Q., et al. Circulation research 115, 488-492(2014)]. Application of in vivo editing to other organ systems arenecessary to prove that this strategy is widely applicable. Currently,efforts to optimize both viral and non-viral vectors are underway toexpand the range of disorders that can be treated with this mode oftherapy [Kotterman, M. A. & Schaffer, D. V. Nature reviews. Genetics 15,445-451 (2014); Yin, H., et al. Nature reviews. Genetics 15, 541-555(2014)]. As discussed herein, the skilled person is positioned from theteachings herein and the knowledge in the art, e.g., Yin to addresshereditary tyrosinemia with a particle-containing HDR template and aCRISPR-Cas (C2c1 or C2c3) system that targets the mutation.

Targeted deletion, therapeutic applications: Targeted deletion of genesmay be preferred. Preferred are, therefore, genes involved inimmunodeficiency disorder, hematologic condition, or genetic lysosomalstorage disease, e.g., Hemophilia B, SCID, SCID-X1, ADA-SCID, Hereditarytyrosinemia, β-thalassemia, X-linked CGD, Wiskott-Aldrich syndrome,Fanconi anemia, adrenoleukodystrophy (ALD), metachromatic leukodystrophy(MLD), HIV/AIDS, other metabolic disorders, genes encoding mis-foldedproteins involved in diseases, genes leading to loss-of-functioninvolved in diseases; generally, mutations that can be targeted in anHSC, using any herein-discussed delivery system, with the particlesystem considered advantageous.

In the present invention, the immunogenicity of the CRISPR enzyme inparticular may be reduced following the approach first set out in Tangriet al with respect to erythropoietin and subsequently developed.Accordingly, directed evolution or rational design may be used to reducethe immunogenicity of the CRISPR enzyme (for instance a C2c1 or C2c3) inthe host species (human or other species).

Genome editing: The CRISPR/Cas (C2c1 or C2c3) systems of the presentinvention can be used to correct genetic mutations that were previouslyattempted with limited success using TALEN and ZFN and lentiviruses,including as herein discussed; see also WO2013163628.

Treating disease of the brian, central nervous and immune systems

The present invention also contemplates delivering the CRISPR-Cas systemto the brain or neurons. For example, RNA interference (RNAi) offerstherapeutic potential for this disorder by reducing the expression ofHT, the disease-causing gene of Huntington's disease (see, e.g., McBrideet al., Molecular Therapy vol. 19 no. 12 Dec. 2011, pp. 2152-2162),therefore Applicant postulates that it may be used and/or adapted to theCRISPR-Cas system. The CRISPR-Cas system may be generated using analgorithm to reduce the off-targeting potential of antisense sequences.The CRISPR-Cas sequences may target either a sequence in exon 52 ofmouse, rhesus or human huntingtin and expressed in a viral vector, suchas AAV. Animals, including humans, may be injected with about threemicroinjections per hemisphere (six injections total): the first 1 mmrostral to the anterior commissure (12 μl) and the two remaininginjections (12 μl and 10 μl, respectively) spaced 3 and 6 mm caudal tothe first injection with 1e12 vg/ml of AAV at a rate of about 1μl/minute, and the needle was left in place for an additional 5 minutesto allow the injectate to diffuse from the needle tip.

DiFiglia et al. (PNAS, Oct. 23, 2007, vol. 104, no. 43, 17204-17209)observed that single administration into the adult striatum of an siRNAtargeting Htt can silence mutant Htt, attenuate neuronal pathology, anddelay the abnormal behavioral phenotype observed in a rapid-onset, viraltransgenic mouse model of HD. DiFiglia injected mice intrastriatallywith 2 μl of Cy3-labeled cc-siRNA-Htt or unconjugated siRNA-Htt at 10μM. A similar dosage of CRISPR Cas targeted to Htt may be contemplatedfor humans in the present invention, for example, about 5-10 ml of 10 sMCRISPR Cas targeted to Htt may be injected intrastriatally.

In another example, Boudreau et al. (Molecular Therapy vol. 17 no. 6Jun. 2009) injects 5 μl of recombinant AAV serotype 2/1 vectorsexpressing htt-specific RNAi virus (at 4×10¹² viral genomes/ml) into thestriatum. A similar dosage of CRISPR Cas targeted to Htt may becontemplated for humans in the present invention, for example, about10-20 ml of 4×10¹² viral genomes/ml) CRISPR Cas targeted to Htt may beinjected intrastriatally.

In another example, a CRISPR Cas targeted to HTT may be administeredcontinuously (see, e.g., Yu et al., Cell 150, 895-908, Aug. 31, 2012).Yu et al. utilizes osmotic pumps delivering 0.25 ml/hr (Model 2004) todeliver 300 mg/day of ss-siRNA or phosphate-buffered saline (PBS) (SigmaAldrich) for 28 days, and pumps designed to deliver 0.5 μl/hr (Model2002) were used to deliver 75 mg/day of the positive control MOE ASO for14 days. Pumps (Durect Corporation) were filled with ss-siRNA or MOEdiluted in sterile PBS and then incubated at 37 C for 24 or 48 (Model2004) hours prior to implantation. Mice were anesthetized with 2.5%isoflurane, and a midline incision was made at the base of the skull.Using stereotaxic guides, a cannula was implanted into the right lateralventricle and secured with Loctite adhesive. A catheter attached to anAlzet osmotic mini pump was attached to the cannula, and the pump wasplaced subcutaneously in the midscapular area. The incision was closedwith 5.0 nylon sutures. A similar dosage of CRISPR Cas targeted to Httmay be contemplated for humans in the present invention, for example,about 500 to 1000 g/day CRISPR Cas targeted to Htt may be administered.

In another example of continuous infusion, Stiles et al. (ExperimentalNeurology 233 (2012) 463-471) implanted an intraparenchymal catheterwith a titanium needle tip into the right putamen. The catheter wasconnected to a SynchroMed® II Pump (Medtronic Neurological, Minneapolis,Minn.) subcutaneously implanted in the abdomen. After a 7 day infusionof phosphate buffered saline at 6 L/day, pumps were re-filled with testarticle and programmed for continuous delivery for 7 days. About 2.3 to11.52 mg/d of siRNA were infused at varying infusion rates of about 0.1to 0.5 L/min. A similar dosage of CRISPR Cas targeted to Htt may becontemplated for humans in the present invention, for example, about 20to 200 mg/day CRISPR Cas targeted to Htt may be administered. In anotherexample, the methods of US Patent Publication No. 20130253040 assignedto Sangamo may also be also be adapted from TALES to the nucleicacid-targeting system of the present invention for treating Huntington'sDisease.

In another example, the methods of US Patent Publication No. 20130253040(WO2013130824) assigned to Sangamo may also be also be adapted fromTALES to the CRISPR Cas system of the present invention for treatingHuntington's Disease.

WO2015089354 A1 in the name of The Broad Institute et al., herebyincorporated by reference, describes a targets for Huntington's Disease(HP). Possible target genes of CRISPR complex in regard to Huntington'sDisease: PRKCE; IGF1; EP300; RCOR1; PRKCZ; HDAC4; and TGM2. Accordingly,one or more of PRKCE; IGF1; EP300; RCOR1; PRKCZ; HDAC4; and TGM2 may beselected as targets for Huntington's Disease in some embodiments of thepresent invention.

Other trinucleotide repeat disorders. These may include any of thefollowing: Category I includes Huntington's disease (HD) and thespinocerebellar ataxias; Category II expansions are phenotypicallydiverse with heterogeneous expansions that are generally small inmagnitude, but also found in the exons of genes; and Category IIIincludes fragile X syndrome, myotonic dystrophy, two of thespinocerebellar ataxias, juvenile myoclonic epilepsy, and Friedreich'sataxia.

A further aspect of the invention relates to utilizing the CRISPR-Cassystem for correcting defects in the EMP2A and EMP2B genes that havebeen identified to be associated with Lafora disease. Lafora disease isan autosomal recessive condition which is characterized by progressivemyoclonus epilepsy which may start as epileptic seizures in adolescence.A few cases of the disease may be caused by mutations in genes yet to beidentified. The disease causes seizures, muscle spasms, difficultywalking, dementia, and eventually death. There is currently no therapythat has proven effective against disease progression. Other geneticabnormalities associated with epilepsy may also be targeted by theCRISPR-Cas system and the underlying genetics is further described inGenetics of Epilepsy and Genetic Epilepsies, edited by GiulianoAvanzini, Jeffrey L. Noebels, Mariani Foundation PaediatricNeurology:20; 2009).

The methods of US Patent Publication No. 20110158957 assigned to SangamoBioSciences, Inc. involved in inactivating T cell receptor (TCR) genesmay also be modified to the CRISPR Cas system of the present invention.In another example, the methods of US Patent Publication No. 20100311124assigned to Sangamo BioSciences, Inc. and US Patent Publication No.20110225664 assigned to Cellectis, which are both involved ininactivating glutamine synthetase gene expression genes may also bemodified to the CRISPR Cas system of the present invention.

Delivery options for the brain include encapsulation of CRISPR enzymeand guide RNA in the form of either DNA or RNA into liposomes andconjugating to molecular Trojan horses for trans-blood brain barrier(BBB) delivery. Molecular Trojan horses have been shown to be effectivefor delivery of B-gal expression vectors into the brain of non-humanprimates. The same approach can be used to delivery vectors containingCRISPR enzyme and guide RNA. For instance, Xia C F and Boado R J,Pardridge W M (“Antibody-mediated targeting of siRNA via the humaninsulin receptor using avidin-biotin technology.” Mol Pharm. 2009May-June; 6(3):747-51. doi: 10.1021/mp800194) describes how delivery ofshort interfering RNA (siRNA) to cells in culture, and in vivo, ispossible with combined use of a receptor-specific monoclonal antibody(mAb) and avidin-biotin technology. The authors also report that becausethe bond between the targeting mAb and the siRNA is stable withavidin-biotin technology, and RNAi effects at distant sites such asbrain are observed in vivo following an intravenous administration ofthe targeted siRNA.

Zhang et al. (Mol Ther. 2003 January; 7(1):11-8.)) describe howexpression plasmids encoding reporters such as luciferase wereencapsulated in the interior of an “artificial virus” comprised of an 85nm pegylated immunoliposome, which was targeted to the rhesus monkeybrain in vivo with a monoclonal antibody (MAb) to the human insulinreceptor (HIR). The HIRMAb enables the liposome carrying the exogenousgene to undergo transcytosis across the blood-brain barrier andendocytosis across the neuronal plasma membrane following intravenousinjection. The level of luciferase gene expression in the brain was50-fold higher in the rhesus monkey as compared to the rat. Widespreadneuronal expression of the beta-galactosidase gene in primate brain wasdemonstrated by both histochemistry and confocal microscopy. The authorsindicate that this approach makes feasible reversible adult transgenicsin 24 hours. Accordingly, the use of immunoliposome is preferred. Thesemay be used in conjunction with antibodies to target specific tissues orcell surface proteins.

Alzheimer's Disease

US Patent Publication No. 20110023153, describes use of zinc fingernucleases to genetically modify cells, animals and proteins associatedwith Alzheimer's Disease. Once modified cells and animals may be furthertested using known methods to study the effects of the targetedmutations on the development and/or progression of AD using measurescommonly used in the study of AD—such as, without limitation, learningand memory, anxiety, depression, addiction, and sensory motor functionsas well as assays that measure behavioral, functional, pathological,metabolic and biochemical function.

The present disclosure comprises editing of any chromosomal sequencesthat encode proteins associated with AD. The AD-related proteins aretypically selected based on an experimental association of theAD-related protein to an AD disorder. For example, the production rateor circulating concentration of an AD-related protein may be elevated ordepressed in a population having an AD disorder relative to a populationlacking the AD disorder. Differences in protein levels may be assessedusing proteomic techniques including but not limited to Western blot,immunohistochemical staining, enzyme linked immunosorbent assay (ELISA),and mass spectrometry. Alternatively, the AD-related proteins may beidentified by obtaining gene expression profiles of the genes encodingthe proteins using genomic techniques including but not limited to DNAmicroarray analysis, serial analysis of gene expression (SAGE), andquantitative real-time polymerase chain reaction (Q-PCR).

Examples of Alzheimer's disease associated proteins may include the verylow density lipoprotein receptor protein (VLDLR) encoded by the VLDLRgene, the ubiquitin-like modifier activating enzyme 1 (UBA1) encoded bythe UBA1 gene, or the NEDD8-activating enzyme El catalytic subunitprotein (UBEIC) encoded by the UBA3 gene, for example.

By way of non-limiting example, proteins associated with AD include butare not limited to the proteins listed as follows: Chromosomal SequenceEncoded Protein ALAS2 Delta-aminolevulinate synthase 2 (ALAS2) ABCA1ATP-binding cassette transporter (ABCA1) ACE Angiotensin I-convertingenzyme (ACE) APOE Apolipoprotein E precursor (APOE) APP amyloidprecursor protein (APP) AQP1 aquaporin 1 protein (AQP1) BIN1 Mycbox-dependent-interacting protein 1 or bridging integrator 1 protein(BIN1) BDNF brain-derived neurotrophic factor (BDNF) BTNL8Butyrophilin-like protein 8 (BTNL8) C1ORF49 chromosome 1 open readingframe 49 CDH4 Cadherin-4 CHRNB2 Neuronal acetylcholine receptor subunitbeta-2 CKLFSF2 CKLF-like MARVEL transmembrane domain-containing protein2 (CKLFSF2) CLEC4E C-type lectin domain family 4, member e (CLEC4E) CLUclusterin protein (also known as apolipoprotein J) CR1 Erythrocytecomplement receptor 1 (CR1, also known as CD35, C3b/C4b receptor andimmune adherence receptor) CR1L Erythrocyte complement receptor 1 (CR1L)CSF3R granulocyte colony-stimulating factor 3 receptor (CSF3R) CST3Cystatin C or cystatin 3 CYP2C Cytochrome P450 2C DAPK1 Death-associatedprotein kinase 1 (DAPK1) ESR1 Estrogen receptor 1 FCAR Fc fragment ofIgA receptor (FCAR, also known as CD89) FCGR3B Fc fragment of IgG, lowaffinity IIIb, receptor (FCGR3B or CD16b) FFA2 Free fatty acid receptor2 (FFA2) FGA Fibrinogen (Factor I) GAB2 GRB2-associated-binding protein2 (GAB2) GAB2 GRB2-associated-binding protein 2 (GAB2) GALP Galanin-likepeptide GAPDHS Glyceraldehyde-3-phosphate dehydrogenase, spermatogenic(GAPDHS) GMPB GMBP HP Haptoglobin (HP) HTR7 5-hydroxytryptamine(serotonin) receptor 7 (adenylate cyclase-coupled) IDE Insulin degradingenzyme IF127 IF127 IFI6 Interferon, alpha-inducible protein 6 (IFI6)IFIT2 Interferon-induced protein with tetratricopeptide repeats 2(IFIT2) ILIRN interleukin-1 receptor antagonist (IL-1RA) IL8RAInterleukin 8 receptor, alpha (IL8RA or CD181) IL8RB Interleukin 8receptor, beta (IL8RB) JAG1 Jagged 1 (JAG1) KCNJ15 Potassiuminwardly-rectifying channel, subfamily J, member 15 (KCNJ15) LRP6Low-density lipoprotein receptor-related protein 6 (LRP6) MAPTmicrotubule-associated protein tau (MAPT) MARK4 MAP/microtubuleaffinity-regulating kinase 4 (MARK4) MPHOSPHI M-phase phosphoprotein 1MTHFR 5,10-methylenetetrahydrofolate reductase MX2 Interferon-inducedGTP-binding protein Mx2 NBN Nibrin, also known as NBN NCSTN NicastrinNIACR2 Niacin receptor 2 (NIACR2, also known as GPR109B) NMNAT3nicotinamide nucleotide adenylyltransferase 3 NTM Neurotrimin (or HNT)ORM1 Orosomucoid 1 (ORM1) or Alpha-i-acid glycoprotein 1 P2RY13 P2Ypurinoceptor 13 (P2RY13) PBEF1 Nicotinamide phosphoribosyltransferase(NAmPRTase or Nampt) also known as pre-B-cell colony-enhancing factor 1(PBEF1) or visfatin PCK1 Phosphoenolpyruvate carboxykinase PICALMphosphatidylinositol binding clathrin assembly protein (PICALM) PLAUUrokinase-type plasminogen activator (PLAU) PLXNC1 Plexin C1 (PLXNC1)PRNP Prion protein PSEN1 presenilin 1 protein (PSEN1) PSEN2 presenilin 2protein (PSEN2) PTPRA protein tyrosine phosphatase receptor type Aprotein (PTPRA) RALGPS2 Ral GEF with PH domain and SH3 binding motif 2(RALGPS2) RGSL2 regulator of G-protein signaling like 2 (RGSL2) SELENBP1Selenium binding protein 1 (SELNBP1) SLC25A37 Mitoferrin-1 SORL1sortilin-related receptor L(DLR class) A repeats-containing protein(SORL1) TF Transferrin TFAM Mitochondrial transcription factor A TNFTumor necrosis factor TNFRSF10C Tumor necrosis factor receptorsuperfamily member 10C (TNFRSF10C) TNFSF10 Tumor necrosis factorreceptor superfamily, (TRAIL) member 10a (TNFSF10) UBA1 ubiquitin-likemodifier activating enzyme 1 (UBA1) UBA3 NEDD8-activating enzyme Elcatalytic subunit protein (UBEIC) UBB ubiquitin B protein (UBB) UBQLN1Ubiquilin-1 UCHL1 ubiquitin carboxyl-terminal esterase L1 protein(UCHL1) UCHL3 ubiquitin carboxyl-terminal hydrolase isozyme L3 protein(UCHL3) VLDLR very low density lipoprotein receptor protein (VLDLR).

In exemplary embodiments, the proteins associated with AD whosechromosomal sequence is edited may be the very low density lipoproteinreceptor protein (VLDLR) encoded by the VLDLR gene, the ubiquitin-likemodifier activating enzyme 1 (UBA1) encoded by the UBAl gene, theNEDD8-activating enzyme El catalytic subunit protein (UBEIC) encoded bythe UBA3 gene, the aquaporin 1 protein (AQP1) encoded by the AQP1 gene,the ubiquitin carboxyl-terminal esterase L1 protein (UCHL1) encoded bythe UCHL1 gene, the ubiquitin carboxyl-terminal hydrolase isozyme L3protein (UCHL3) encoded by the UCHL3 gene, the ubiquitin B protein (UBB)encoded by the UBB gene, the microtubule-associated protein tau (MAPT)encoded by the MAPT gene, the protein tyrosine phosphatase receptor typeA protein (PTPRA) encoded by the PTPRA gene, the phosphatidylinositolbinding clathrin assembly protein (PICALM) encoded by the PICALM gene,the clusterin protein (also known as apoplipoprotein J) encoded by theCLU gene, the presenilin 1 protein encoded by the PSEN1 gene, thepresenilin 2 protein encoded by the PSEN2 gene, the sortilin-relatedreceptor L(DLR class) A repeats-containing protein (SORL1) proteinencoded by the SORL1 gene, the amyloid precursor protein (APP) encodedby the APP gene, the Apolipoprotein E precursor (APOE) encoded by theAPOE gene, or the brain-derived neurotrophic factor (BDNF) encoded bythe BDNF gene. In an exemplary embodiment, the genetically modifiedanimal is a rat, and the edited chromosomal sequence encoding theprotein associated with AD is as as follows: APP amyloid precursorprotein (APP) NM_019288 AQP1 aquaporin 1 protein (AQP1) NM_012778 BDNFBrain-derived neurotrophic factor NM_012513 CLU clusterin protein (alsoknown as NM_053021 apoplipoprotein J) MAPT microtubule-associatedprotein NM_017212 tau (MAPT) PICALM phosphatidylinositol bindingNM_053554 clathrin assembly protein (PICALM) PSEN1 presenilin 1 protein(PSEN1) NM_019163 PSEN2 presenilin 2 protein (PSEN2) NM_031087 PTPRAprotein tyrosine phosphatase NM_012763 receptor type A protein (PTPRA)SORL1 sortilin-related receptor L(DLR NM_053519, class) Arepeats-containing XM_001065506, protein (SORL1) XM_217115 UBAlubiquitin-like modifier activating NM_001014080 enzyme 1 (UBA1) UBA3NEDD8-activating enzyme El NM_057205 catalytic subunit protein (UBE1C)UBB ubiquitin B protein (UBB) NM_138895 UCHL1 ubiquitincarboxyl-terminal NM_017237 esterase L1 protein (UCHL1) UCHL3 ubiquitincarboxyl-terminal NM_001110165 hydrolase isozyme L3 protein (UCHL3)VLDLR very low density lipoprotein NM_013155 receptor protein (VLDLR)

The animal or cell may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12,13, 14, 15 or more disrupted chromosomal sequences encoding a proteinassociated with AD and zero, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15 or more chromosomally integrated sequences encoding a proteinassociated with AD.

The edited or integrated chromosomal sequence may be modified to encodean altered protein associated with AD. A number of mutations inAD-related chromosomal sequences have been associated with AD. Forinstance, the V7171 (i.e. valine at position 717 is changed toisoleucine) missense mutation in APP causes familial AD. Multiplemutations in the presenilin-1 protein, such as H163R (i.e. histidine atposition 163 is changed to arginine), A246E (i.e. alanine at position246 is changed to glutamate), L286V (i.e. leucine at position 286 ischanged to valine) and C410Y (i.e. cysteine at position 410 is changedto tyrosine) cause familial Alzheimer's type 3. Mutations in thepresenilin-2 protein, such as N141 I (i.e. asparagine at position 141 ischanged to isoleucine), M239V (i.e. methionine at position 239 ischanged to valine), and D439A (i.e. aspartate at position 439 is changedto alanine) cause familial Alzheimer's type 4. Other associations ofgenetic variants in AD-associated genes and disease are known in theart. See, for example, Waring et al. (2008) Arch. Neurol. 65:329-334,the disclosure of which is incorporated by reference herein in itsentirety.

Secretase Disorders

US Patent Publication No. 20110023146, describes use of zinc fingernucleases to genetically modify cells, animals and proteins associatedwith secretase-associated disorders. Secretases are essential forprocessing pre-proteins into their biologically active forms. Defects invarious components of the secretase pathways contribute to manydisorders, particularly those with hallmark amyloidogenesis or amyloidplaques, such as Alzheimer's disease (AD).

A secretase disorder and the proteins associated with these disordersare a diverse set of proteins that effect susceptibility for numerousdisorders, the presence of the disorder, the severity of the disorder,or any combination thereof. The present disclosure comprises editing ofany chromosomal sequences that encode proteins associated with asecretase disorder. The proteins associated with a secretase disorderare typically selected based on an experimental association of thesecretase-related proteins with the development of a secretase disorder.For example, the production rate or circulating concentration of aprotein associated with a secretase disorder may be elevated ordepressed in a population with a secretase disorder relative to apopulation without a secretase disorder. Differences in protein levelsmay be assessed using proteomic techniques including but not limited toWestern blot, immunohistochemical staining, enzyme linked immunosorbentassay (ELISA), and mass spectrometry. Alternatively, the proteinassociated with a secretase disorder may be identified by obtaining geneexpression profiles of the genes encoding the proteins using genomictechniques including but not limited to DNA microarray analysis, serialanalysis of gene expression (SAGE), and quantitative real-timepolymerase chain reaction (Q-PCR).

By way of non-limiting example, proteins associated with a secretasedisorder include PSENEN (presenilin enhancer 2 homolog (C. elegans)),CTSB (cathepsin B), PSEN1 (presenilin 1), APP (amyloid beta (A4)precursor protein), APH1B (anterior pharynx defective 1 homolog B (C.elegans)), PSEN2 (presenilin 2 (Alzheimer disease 4)), BACE1 (beta-siteAPP-cleaving enzyme 1), ITM2B (integral membrane protein 2B), CTSD(cathepsin D), NOTCH1 (Notch homolog 1, translocation-associated(Drosophila)), TNF (tumor necrosis factor (TNF superfamily, member 2)),INS (insulin), DYT10 (dystonia 10), ADAM17 (ADAM metallopeptidase domain17), APOE (apolipoprotein E), ACE (angiotensin I converting enzyme(peptidyl-dipeptidase A) 1), STN (statin), TP53 (tumor protein p53), I16(interleukin 6 (interferon, beta 2)), NGFR (nerve growth factor receptor(TNFR superfamily, member 16)), ILIB (interleukin 1, beta), ACHE(acetylcholinesterase (Yt blood group)), CTNNB1 (catenin(cadherin-associated protein), beta 1, 88 kDa), IGF1 (insulin-likegrowth factor 1 (somatomedin C)), IFNG (interferon, gamma), NRG1(neuregulin 1), CASP3 (caspase 3, apoptosis-related cysteine peptidase),MAPK1 (mitogen-activated protein kinase 1), CDH1 (cadherin 1, type 1,E-cadherin (epithelial)), APBB1 (amyloid beta (A4) precursorprotein-binding, family B, member 1 (Fe65)), HMGCR(3-hydroxy-3-methylglutaryl-Coenzyme A reductase), CREB1 (cAMPresponsive element binding protein 1), PTGS2 (prostaglandin-endoperoxidesynthase 2 (prostaglandin G/H synthase and cyclooxygenase)), HES1 (hairyand enhancer of split 1, (Drosophila)), CAT (catalase), TGFB1(transforming growth factor, beta 1), ENO2 (enolase 2 (gamma,neuronal)), ERBB4 (v-erb-a erythroblastic leukemia viral oncogenehomolog 4 (avian)), TRAPPC10 (trafficking protein particle complex 10),MAOB (monoamine oxidase B), NGF (nerve growth factor (betapolypeptide)), MMP12 (matrix metallopeptidase 12 (macrophage elastase)),JAG1 (jagged 1 (Alagille syndrome)), CD40LG (CD40 ligand), PPARG(peroxisome proliferator-activated receptor gamma), FGF2 (fibroblastgrowth factor 2 (basic)), IL3 (interleukin 3 (colony-stimulating factor,multiple)), LRP1 (low density lipoprotein receptor-related protein 1),NOTCH4 (Notch homolog 4 (Drosophila)), MAPK8 (mitogen-activated proteinkinase 8), PREP (prolyl endopeptidase), NOTCH3 (Notch homolog 3(Drosophila)), PRNP (prion protein), CTSG (cathepsin G), EGF (epidermalgrowth factor (beta-urogastrone)), REN (renin), CD44 (CD44 molecule(Indian blood group)), SELP (selectin P (granule membrane protein 140kDa, antigen CD62)), GHR (growth hormone receptor), ADCYAP1 (adenylatecyclase activating polypeptide 1 (pituitary)), INSR (insulin receptor),GFAP (glial fibrillary acidic protein), MMP3 (matrix metallopeptidase 3(stromelysin 1, progelatinase)), MAPK10 (mitogen-activated proteinkinase 10), SP1 (Sp1 transcription factor), MYC (v-myc myelocytomatosisviral oncogene homolog (avian)), CTSE (cathepsin E), PPARA (peroxisomeproliferator-activated receptor alpha), JUN (jun oncogene), TIMP1 (TIMPmetallopeptidase inhibitor 1), IL5 (interleukin 5 (colony-stimulatingfactor, eosinophil)), ILIA (interleukin 1, alpha), MMP9 (matrixmetallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IVcollagenase)), HTR4 (5-hydroxytryptamine (serotonin) receptor 4), HSPG2(heparan sulfate proteoglycan 2), KRAS (v-Ki-ras2 Kirsten rat sarcomaviral oncogene homolog), CYCS (cytochrome c, somatic), SMG1 (SMG1homolog, phosphatidylinositol 3-kinase-related kinase (C. elegans)),ILIR1 (interleukin 1 receptor, type I), PROK (prokineticin 1), MAPK3(mitogen-activated protein kinase 3), NTRK1 (neurotrophic tyrosinekinase, receptor, type 1), IL13 (interleukin 13), MME (membranemetallo-endopeptidase), TKT (transketolase), CXCR2 (chemokine (C—X—Cmotif) receptor 2), IGF1R (insulin-like growth factor 1 receptor), RARA(retinoic acid receptor, alpha), CREBBP (CREB binding protein), PTGS1(prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase andcyclooxygenase)), GALT (galactose-1-phosphate uridylyltransferase),CHRM1 (cholinergic receptor, muscarinic 1), ATXN1 (ataxin 1), PAWR(PRKC, apoptosis, WT1, regulator), NOTCH2 (Notch homolog 2(Drosophila)), M6PR (mannose-6-phosphate receptor (cation dependent)),CYP46A1 (cytochrome P450, family 46, subfamily A, polypeptide 1), CSNK1D (casein kinase 1, delta), MAPK14 (mitogen-activated protein kinase14), PRG2 (proteoglycan 2, bone marrow (natural killer cell activator,eosinophil granule major basic protein)), PRKCA (protein kinase C,alpha), Li CAM (L1 cell adhesion molecule), CD40 (CD40 molecule, TNFreceptor superfamily member 5), NR1I2 (nuclear receptor subfamily 1,group I, member 2), JAG2 (jagged 2), CTNND1 (catenin(cadherin-associated protein), delta 1), CDH2 (cadherin 2, type 1,N-cadherin (neuronal)), CMAi (chymase 1, mast cell), SORT1 (sortilin 1),DLK1 (delta-like 1 homolog (Drosophila)), THEM4 (thioesterasesuperfamily member 4), JUP (junction plakoglobin), CD46 (CD46 molecule,complement regulatory protein), CCL11 (chemokine (C-C motif) ligand 11),CAV3 (caveolin 3), RNASE3 (ribonuclease, RNase A family, 3 (eosinophilcationic protein)), HSPA8 (heat shock 70 kDa protein 8), CASP9 (caspase9, apoptosis-related cysteine peptidase), CYP3A4 (cytochrome P450,family 3, subfamily A, polypeptide 4), CCR3 (chemokine (C—C motif)receptor 3), TFAP2A (transcription factor AP-2 alpha (activatingenhancer binding protein 2 alpha)), SCP2 (sterol carrier protein 2),CDK4 (cyclin-dependent kinase 4), HIF1A (hypoxia inducible factor 1,alpha subunit (basic helix-loop-helix transcription factor)), TCF7L2(transcription factor 7-like 2 (T-cell specific, HMG-box)), ILIR2(interleukin 1 receptor, type II), B3GALTL (beta1,3-galactosyltransferase-like), MDM2 (Mdm2 p53 binding protein homolog(mouse)), RELA (v-rel reticuloendotheliosis viral oncogene homolog A(avian)), CASP7 (caspase 7, apoptosis-related cysteine peptidase), IDE(insulin-degrading enzyme), FABP4 (fatty acid binding protein 4,adipocyte), CASK (calcium/calmodulin-dependent serine protein kinase(MAGUK family)), ADCYAP1R1 (adenylate cyclase activating polypeptide 1(pituitary) receptor type I), ATF4 (activating transcription factor 4(tax-responsive enhancer element B67)), PDGFA (platelet-derived growthfactor alpha polypeptide), C21 or f33 (chromosome 21 open reading frame33), SCG5 (secretogranin V (7B2 protein)), RNF123 (ring finger protein123), NFKB1 (nuclear factor of kappa light polypeptide gene enhancer inB-cells 1), ERBB2 (v-erb-b2 erythroblastic leukemia viral oncogenehomolog 2, neuro/glioblastoma derived oncogene homolog (avian)), CAV1(caveolin 1, caveolae protein, 22 kDa), MMP7 (matrix metallopeptidase 7(matrilysin, uterine)), TGFA (transforming growth factor, alpha), RXRA(retinoid X receptor, alpha), STX1A (syntaxin 1A (brain)), PSMC4(proteasome (prosome, macropain) 26S subunit, ATPase, 4), P2RY2(purinergic receptor P2Y, G-protein coupled, 2), TNFRSF21 (tumornecrosis factor receptor superfamily, member 21), DLG1 (discs, largehomolog 1 (Drosophila)), NUMBL (numb homolog (Drosophila)-like), SPN(sialophorin), PLSCR1 (phospholipid scramblase 1), UBQLN2 (ubiquilin 2),UBQLN1 (ubiquilin 1), PCSK7 (proprotein convertase subtilisin/kexin type7), SPON1 (spondin 1, extracellular matrix protein), SILV (silverhomolog (mouse)), QPCT (glutaminyl-peptide cyclotransferase), HESS(hairy and enhancer of split 5 (Drosophila)), GCC1 (GRIP and coiled-coildomain containing 1), and any combination thereof.

The genetically modified animal or cell may comprise 1, 2, 3, 4, 5, 6,7, 8, 9, 10 or more disrupted chromosomal sequences encoding a proteinassociated with a secretase disorder and zero, 1, 2, 3, 4, 5, 6, 7, 8,9, 10 or more chromosomally integrated sequences encoding a disruptedprotein associated with a secretase disorder.

ALS

US Patent Publication No. 20110023144, describes use of zinc fingernucleases to genetically modify cells, animals and proteins associatedwith amyotrophic lateral sclerosis (ALS) disease. ALS is characterizedby the gradual steady degeneration of certain nerve cells in the braincortex, brain stem, and spinal cord involved in voluntary movement.

Motor neuron disorders and the proteins associated with these disordersare a diverse set of proteins that effect susceptibility for developinga motor neuron disorder, the presence of the motor neuron disorder, theseverity of the motor neuron disorder or any combination thereof. Thepresent disclosure comprises editing of any chromosomal sequences thatencode proteins associated with ALS disease, a specific motor neurondisorder. The proteins associated with ALS are typically selected basedon an experimental association of ALS-related proteins to ALS. Forexample, the production rate or circulating concentration of a proteinassociated with ALS may be elevated or depressed in a population withALS relative to a population without ALS. Differences in protein levelsmay be assessed using proteomic techniques including but not limited toWestern blot, immunohistochemical staining, enzyme linked immunosorbentassay (ELISA), and mass spectrometry. Alternatively, the proteinsassociated with ALS may be identified by obtaining gene expressionprofiles of the genes encoding the proteins using genomic techniquesincluding but not limited to DNA microarray analysis, serial analysis ofgene expression (SAGE), and quantitative real-time polymerase chainreaction (Q-PCR).

By way of non-limiting example, proteins associated with ALS include butare not limited to the following proteins: SOD1 superoxide dismutase 1,ALS3 amyotrophic lateral soluble sclerosis 3 SETX senataxin ALS5amyotrophic lateral sclerosis 5 FUS fused in sarcoma ALS7 amyotrophiclateral sclerosis 7 ALS2 amyotrophic lateral DPP6 Dipeptidyl-peptidase 6sclerosis 2 NEFH neurofilament, heavy PTGS1 prostaglandin-polypeptideendoperoxide synthase 1 SLC1A2 solute carrier family 1 TNFRSF10B tumornecrosis factor (glial high affinity receptor superfamily, glutamatetransporter), member 10b member 2 PRPH peripherin HSP90AAI heat shockprotein 90 kDa alpha (cytosolic), class A member 1 GRIA2 glutamatereceptor, IFNG interferon, gamma ionotropic, AMPA 2 S100B S100 calciumbinding FGF2 fibroblast growth factor 2 protein B AOX1 aldehyde oxidase1 CS citrate synthase TARDBP TAR DNA binding protein TXN thioredoxinRAPH1 Ras association MAP3K5 mitogen-activated protein (RaIGDS/AF-6) andkinase 5 pleckstrin homology domains 1 NBEAL1 neurobeachin-like 1 GPX1glutathione peroxidase 1 ICA1L islet cell autoantigen RAC1 ras-relatedC3 botulinum 1.69 kDa-like toxin substrate 1 MAPT microtubule-associatedITPR2 inositol 1,4,5-protein tau triphosphate receptor, type 2 ALS2CR4amyotrophic lateral GLS glutaminase sclerosis 2 (juvenile) chromosomeregion, candidate 4 ALS2CR8 amyotrophic lateral CNTFR ciliaryneurotrophic factor sclerosis 2 (juvenile) receptor chromosome region,candidate 8 ALS2CR11 amyotrophic lateral FOLH1 folate hydrolase 1sclerosis 2 (juvenile) chromosome region, candidate 11 FAM117B familywith sequence P4HB prolyl 4-hydroxylase, similarity 117, member B betapolypeptide CNTF ciliary neurotrophic factor SQSTM1 sequestosome 1STRADB STE20-related kinase NAIP NLR family, apoptosis adaptor betainhibitory protein YWHAQ tyrosine 3-SLC33A1 solute carrier family 33monooxygenase/tryptophan (acetyl-CoA transporter), an 5-monooxygenasemember 1 activation protein, theta polypeptide TRAK2 traffickingprotein, FIG. 4 homolog, SAC1 kinesin binding 2 lipid phosphatase domaincontaining NIF3L1 NIF3 NGG1 interacting INA internexin neuronal factor3-like 1 intermediate filament protein, alpha PARD3B par-3 partitioningCOX8A cytochrome c oxidase defective 3 homolog B subunit VIIIA CDK15cyclin-dependent kinase HECW1 HECT, C2 and WW 15 domain containing E3ubiquitin protein ligase 1 NOSi nitric oxide synthase 1 MET metproto-oncogene SOD2 superoxide dismutase 2, HSPB1 heat shock 27 kDamitochondrial protein 1 NEFL neurofilament, light CTSB cathepsin Bpolypeptide ANG angiogenin, HSPA8 heat shock 70 kDa ribonuclease, RNaseA protein 8 family, 5 VAPB VAMP (vesicle-ESR1 estrogen receptor 1associated membrane protein)-associated protein B and C SNCA synuclein,alpha HGF hepatocyte growth factor CAT catalase ACTB actin, beta NEFMneurofilament, medium TH tyrosine hydroxylase polypeptide BCL2 B-cellCLL/lymphoma 2 FAS Fas (TNF receptor superfamily, member 6) CASP3caspase 3, apoptosis-CLU clusterin related cysteine peptidase SMN1survival of motor neuron G6PD glucose-6-phosphate 1, telomericdehydrogenase BAX BCL2-associated X HSF1 heat shock transcriptionprotein factor 1 RNF19A ring finger protein 19A JUN jun oncogeneALS2CR12 amyotrophic lateral HSPA5 heat shock 70 kDa sclerosis 2(juvenile) protein 5 chromosome region, candidate 12 MAPK14mitogen-activated protein IL10 interleukin 10 kinase 14 APEX1 APEXnuclease TXNRD1 thioredoxin reductase 1 (multifunctional DNA repairenzyme) 1 NOS2 nitric oxide synthase 2, TIMP1 TIMP metallopeptidaseinducible inhibitor 1 CASP9 caspase 9, apoptosis-XIAP X-linked inhibitorof related cysteine apoptosis peptidase GLG1 golgi glycoprotein 1 EPOerythropoietin VEGFA vascular endothelial ELN elastin growth factor AGDNF glial cell derived NFE2L2 nuclear factor (erythroid-neurotrophicfactor derived 2)-like 2 SLC6A3 solute carrier family 6 HSPA4 heat shock70 kDa (neurotransmitter protein 4 transporter, dopamine), member 3 APOEapolipoprotein E PSMB8 proteasome (prosome, macropain) subunit, betatype, 8 DCTN1 dynactin 1 TIMP3 TIMP metallopeptidase inhibitor 3 KIFAP3kinesin-associated SLC1A1 solute carrier family 1 protein 3(neuronal/epithelial high affinity glutamate transporter, system Xag),member 1 SMN2 survival of motor neuron CCNC cyclin C 2, centromeric MPP4membrane protein, STUB1 STIP1 homology and U-palmitoylated 4 boxcontaining protein 1 ALS2 amyloid beta (A4) PRDX6 peroxiredoxin 6precursor protein SYP synaptophysin CABIN1 calcineurin binding protein 1CASP1 caspase 1, apoptosis-GART phosphoribosylglycinamide relatedcysteine formyltransferase, peptidase phosphoribosylglycinamidesynthetase, phosphoribosylaminoimidazole synthetase CDK5cyclin-dependent kinase 5 ATXN3 ataxin 3 RTN4 reticulon 4 C1QBcomplement component 1, q subcomponent, B chain VEGFC nerve growthfactor HTT huntingtin receptor PARK7 Parkinson disease 7 XDH xanthinedehydrogenase GFAP glial fibrillary acidic MAP2 microtubule-associatedprotein protein 2 CYCS cytochrome c, somatic FCGR3B Fc fragment of IgG,low affinity IIIb, CCS copper chaperone for UBL5 ubiquitin-like 5superoxide dismutase MMP9 matrix metallopeptidase SLC18A3 solute carrierfamily 18 9 ((vesicular acetylcholine), member 3 TRPM7 transientreceptor HSPB2 heat shock 27 kDa potential cation channel, protein 2subfamily M, member 7 AKT1 v-akt murine thymoma DERL1 Derl-like domainfamily, viral oncogene homolog 1 member 1 CCL2 chemokine (C-C motif)NGRN neugrin, neurite ligand 2 outgrowth associated GSR glutathionereductase TPPP3 tubulin polymerization-promoting protein family member 3APAF1 apoptotic peptidase BTBD10 BTB (POZ) domain activating factor 1containing 10 GLUD1 glutamate CXCR4 chemokine (C—X—C motif)dehydrogenase 1 receptor 4 SLC1A3 solute carrier family 1 FLT1fms-related tyrosine (glial high affinity glutamate transporter), member3 kinase 1 PON1 paraoxonase 1 AR androgen receptor LIF leukemiainhibitory factor ERBB3 v-erb-b2 erythroblastic leukemia viral oncogenehomolog 3 LGALS1 lectin, galactoside-CD44 CD44 molecule binding,soluble, 1 TP53 tumor protein p53 TLR3 toll-like receptor 3 GRIA1glutamate receptor, GAPDH glyceraldehyde-3-ionotropic, AMPA 1 phosphatedehydrogenase GRIK1 glutamate receptor, DES desmin ionotropic, kainate 1CHAT choline acetyltransferase FLT4 fms-related tyrosine kinase 4 CHMP2Bchromatin modifying BAG1 BCL2-associated protein 2B athanogene MT3metallothionein 3 CHRNA4 cholinergic receptor, nicotinic, alpha 4 GSSglutathione synthetase BAK1 BCL2-antagonist/killer 1 KDR kinase insertdomain GSTP1 glutathione S-transferase receptor (a type III pi 1receptor tyrosine kinase) OGG1 8-oxoguanine DNA IL6 interleukin 6(interferon, glycosylase beta 2).

The animal or cell may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moredisrupted chromosomal sequences encoding a protein associated with ALSand zero, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chromosomally integratedsequences encoding the disrupted protein associated with ALS. Preferredproteins associated with ALS include SOD1 (superoxide dismutase 1), ALS2(amyotrophic lateral sclerosis 2), FUS (fused in sarcoma), TARDBP (TARDNA binding protein), VAGFA (vascular endothelial growth factor A),VAGFB (vascular endothelial growth factor B), and VAGFC (vascularendothelial growth factor C), and any combination thereof.

Autism

US Patent Publication No. 20110023145, describes use of zinc fingernucleases to genetically modify cells, animals and proteins associatedwith autism spectrum disorders (ASD). Autism spectrum disorders (ASDs)are a group of disorders characterized by qualitative impairment insocial interaction and communication, and restricted repetitive andstereotyped patterns of behavior, interests, and activities. The threedisorders, autism, Asperger syndrome (AS) and pervasive developmentaldisorder-not otherwise specified (PDD-NOS) are a continuum of the samedisorder with varying degrees of severity, associated intellectualfunctioning and medical conditions. ASDs are predominantly geneticallydetermined disorders with a heritability of around 90%.

US Patent Publication No. 20110023145 comprises editing of anychromosomal sequences that encode proteins associated with ASD which maybe applied to the CRISPR Cas system of the present invention. Theproteins associated with ASD are typically selected based on anexperimental association of the protein associated with ASD to anincidence or indication of an ASD. For example, the production rate orcirculating concentration of a protein associated with ASD may beelevated or depressed in a population having an ASD relative to apopulation lacking the ASD. Differences in protein levels may beassessed using proteomic techniques including but not limited to Westernblot, immunohistochemical staining, enzyme linked immunosorbent assay(ELISA), and mass spectrometry. Alternatively, the proteins associatedwith ASD may be identified by obtaining gene expression profiles of thegenes encoding the proteins using genomic techniques including but notlimited to DNA microarray analysis, serial analysis of gene expression(SAGE), and quantitative real-time polymerase chain reaction (Q-PCR).

Non limiting examples of disease states or disorders that may beassociated with proteins associated with ASD include autism, Aspergersyndrome (AS), pervasive developmental disorder-not otherwise specified(PDD-NOS), Rett's syndrome, tuberous sclerosis, phenylketonuria,Smith-Lemli-Opitz syndrome and fragile X syndrome. By way ofnon-limiting example, proteins associated with ASD include but are notlimited to the following proteins: ATP10C aminophospholipid-MET METreceptor transporting ATPase tyrosine kinase (ATP10C) BZRAP1 MGLUR5(GRM5) Metabotropic glutamate receptor 5 (MGLUR5) CDH10 Cadherin-10MGLUR6 (GRM6) Metabotropic glutamate receptor 6 (MGLUR6) CDH9 Cadherin-9NLGN1 Neuroligin-1 CNTN4 Contactin-4 NLGN2 Neuroligin-2 CNTNAP2Contactin-associated SEMASA Neuroligin-3 protein-like 2 (CNTNAP2) DHCR77-dehydrocholesterol NLGN4X Neuroligin-4 X-reductase (DHCR7) linkedDOC2A Double C2-like domain-NLGN4Y Neuroligin-4 Y-containing proteinalpha linked DPP6 Dipeptidyl NLGN5 Neuroligin-5 aminopeptidase-likeprotein 6 EN2 engrailed 2 (EN2) NRCAM Neuronal cell adhesion molecule(NRCAM) MDGA2 fragile X mental retardation NRXN1 Neurexin-1 1 (MDGA2)FMR2 (AFF2) AF4/FMR2 family member 2 OR4M2 Olfactory receptor (AFF2) 4M2FOXP2 Forkhead box protein P2 OR4N4 Olfactory receptor (FOXP2) 4N4 FXR1Fragile X mental OXTR oxytocin receptor retardation, autosomal (OXTR)homolog 1 (FXR1) FXR2 Fragile X mental PAH phenylalanine retardation,autosomal hydroxylase (PAH) homolog 2 (FXR2) GABRA1 Gamma-aminobutyricacid PTEN Phosphatase and receptor subunit alpha-1 tensin homologue(GABRA1) (PTEN) GABRA5 GABAA (.gamma.-aminobutyric PTPRZ1 Receptor-typeacid) receptor alpha 5 tyrosine-protein subunit (GABRA5) phosphatasezeta (PTPRZI) GABRB1 Gamma-aminobutyric acid RELN Reelin receptorsubunit beta-1 (GABRB1) GABRB3 GABAA (.gamma.-aminobutyric RPL10 60Sribosomal acid) receptor .beta.3 subunit protein L10 (GABRB3) GABRG1Gamma-aminobutyric acid SEMA5A Semaphorin-5A receptor subunit gamma-1(SEMA5A) (GABRG1) HIRIP3 HIRA-interacting protein 3 SEZ6L2 seizurerelated 6 homolog (mouse)-like 2 HOXA1 Homeobox protein Hox-A1 SHANK3SH3 and multiple (HOXA1) ankyrin repeat domains 3 (SHANK3) IL6Interleukin-6 SHBZRAPI SH3 and multiple ankyrin repeat domains 3(SHBZRAPI) LAMB1 Laminin subunit beta-1 SLC6A4 Serotonin (LAMB1)transporter (SERT) MAPK3 Mitogen-activated protein TAS2R1 Taste receptorkinase 3 type 2 member 1 TAS2R1 MAZ Myc-associated zinc finger TSC1Tuberous sclerosis protein protein 1 MDGA2 MAM domain containing TSC2Tuberous sclerosis glycosylphosphatidylinositol protein 2 anchor 2(MDGA2) MECP2 Methyl CpG binding UBE3A Ubiquitin protein protein 2(MECP2) ligase E3A (UBE3A) MECP2 methyl CpG binding WNT2 Wingless-typeprotein 2 (MECP2) MMTV integration site family, member 2 (WNT2).

The identity of the protein associated with ASD whose chromosomalsequence is edited can and will vary. In preferred embodiments, theproteins associated with ASD whose chromosomal sequence is edited may bethe benzodiazapine receptor (peripheral) associated protein 1 (BZRAP1)encoded by the BZRAP1 gene, the AF4/FMR2 family member 2 protein (AFF2)encoded by the AFF2 gene (also termed MFR2), the fragile X mentalretardation autosomal homolog 1 protein (FXR1) encoded by the FXR1 gene,the fragile X mental retardation autosomal homolog 2 protein (FXR2)encoded by the FXR2 gene, the MAM domain containingglycosylphosphatidylinositol anchor 2 protein (MDGA2) encoded by theMDGA2 gene, the methyl CpG binding protein 2 (MECP2) encoded by theMECP2 gene, the metabotropic glutamate receptor 5 (MGLUR5) encoded bythe MGLUR5-1 gene (also termed GRM5), the neurexin 1 protein encoded bythe NRXN1 gene, or the semaphorin-5A protein (SEMA5A) encoded by theSEMA5A gene. In an exemplary embodiment, the genetically modified animalis a rat, and the edited chromosomal sequence encoding the proteinassociated with ASD is as listed below: BZRAP1 benzodiazepine receptorXM_002727789, (peripheral) associated XM_213427, protein 1 (BZRAP1)XM_002724533, XM_001081125 AFF2 (FMR2) AF4/FMR2 family member 2XM_219832, (AFF2) XM_001054673 FXR1 Fragile X mental NM_001012179retardation, autosomal homolog 1 (FXR1) FXR2 Fragile X mentalNM_001100647 retardation, autosomal homolog 2 (FXR2) MDGA2 MAM domaincontaining NM_199269 glycosylphosphatidylinositol anchor 2 (MDGA2) MECP2Methyl CpG binding NM_022673 protein 2 (MECP2) MGLUR5 Metabotropicglutamate NM_017012 (GRM5) receptor 5 (MGLUR5) NRXN1 Neurexin-1NM_021767 SEMA5A Semaphorin-5A (SEMA5A) NM_001107659.

Trinucleotide Repeat Expansion Disorders

US Patent Publication No. 20110016540, describes use of zinc fingernucleases to genetically modify cells, animals and proteins associatedwith trinucleotide repeat expansion disorders. Trinucleotide repeatexpansion disorders are complex, progressive disorders that involvedevelopmental neurobiology and often affect cognition as well assensori-motor functions.

Trinucleotide repeat expansion proteins are a diverse set of proteinsassociated with susceptibility for developing a trinucleotide repeatexpansion disorder, the presence of a trinucleotide repeat expansiondisorder, the severity of a trinucleotide repeat expansion disorder orany combination thereof. Trinucleotide repeat expansion disorders aredivided into two categories determined by the type of repeat. The mostcommon repeat is the triplet CAG, which, when present in the codingregion of a gene, codes for the amino acid glutamine (Q). Therefore,these disorders are referred to as the polyglutamine (polyQ) disordersand comprise the following diseases: Huntington Disease (HD);Spinobulbar Muscular Atrophy (SBMA); Spinocerebellar Ataxias (SCA types1, 2, 3, 6, 7, and 17); and Dentatorubro-Pallidoluysian Atrophy (DRPLA).The remaining trinucleotide repeat expansion disorders either do notinvolve the CAG triplet or the CAG triplet is not in the coding regionof the gene and are, therefore, referred to as the non-polyglutaminedisorders. The non-polyglutamine disorders comprise Fragile X Syndrome(FRAXA); Fragile XE Mental Retardation (FRAXE); Friedreich Ataxia(FRDA); Myotonic Dystrophy (DM); and Spinocerebellar Ataxias (SCA types8, and 12).

The proteins associated with trinucleotide repeat expansion disordersare typically selected based on an experimental association of theprotein associated with a trinucleotide repeat expansion disorder to atrinucleotide repeat expansion disorder. For example, the productionrate or circulating concentration of a protein associated with atrinucleotide repeat expansion disorder may be elevated or depressed ina population having a trinucleotide repeat expansion disorder relativeto a population lacking the trinucleotide repeat expansion disorder.Differences in protein levels may be assessed using proteomic techniquesincluding but not limited to Western blot, immunohistochemical staining,enzyme linked immunosorbent assay (ELISA), and mass spectrometry.Alternatively, the proteins associated with trinucleotide repeatexpansion disorders may be identified by obtaining gene expressionprofiles of the genes encoding the proteins using genomic techniquesincluding but not limited to DNA microarray analysis, serial analysis ofgene expression (SAGE), and quantitative real-time polymerase chainreaction (Q-PCR).

Non-limiting examples of proteins associated with trinucleotide repeatexpansion disorders include AR (androgen receptor), FMR1 (fragile Xmental retardation 1), HTT (huntingtin), DMPK (dystrophiamyotonica-protein kinase), FXN (frataxin), ATXN2 (ataxin 2), ATN1(atrophin 1), FEN1 (flap structure-specific endonuclease 1), TNRC6A(trinucleotide repeat containing 6A), PABPN1 (poly(A) binding protein,nuclear 1), JPH3 (junctophilin 3), MED15 (mediator complex subunit 15),ATXN1 (ataxin 1), ATXN3 (ataxin 3), TBP (TATA box binding protein),CACNA1A (calcium channel, voltage-dependent, P/Q type, alpha 1Asubunit), ATXN80S (ATXN8 opposite strand (non-protein coding)), PPP2R2B(protein phosphatase 2, regulatory subunit B, beta), ATXN7 (ataxin 7),TNRC6B (trinucleotide repeat containing 6B), TNRC6C (trinucleotiderepeat containing 6C), CELF3 (CUGBP, Elav-like family member 3), MAB21L1(mab-21-like 1 (C. elegans)), MSH2 (mutS homolog 2, colon cancer,nonpolyposis type 1 (E. coli)), TMEM185A (transmembrane protein 185A),SIX5 (SIX homeobox 5), CNPY3 (canopy 3 homolog (zebrafish)), FRAXE(fragile site, folic acid type, rare, fra(X)(q28) E), GNB2 (guaninenucleotide binding protein (G protein), beta polypeptide 2), RPL14(ribosomal protein L14), ATXN8 (ataxin 8), INSR (insulin receptor), TTR(transthyretin), EP400 (E1A binding protein p400), GIGYF2 (GRB10interacting GYF protein 2), OGG1 (8-oxoguanine DNA glycosylase), STC1(stanniocalcin 1), CNDP1 (carnosine dipeptidase 1 (metallopeptidase M20family)), C10orf2 (chromosome 10 open reading frame 2), MAML3mastermind-like 3 (Drosophila), DKC1 (dyskeratosis congenita 1,dyskerin), PAXIP1 (PAX interacting (with transcription-activationdomain) protein 1), CASK (calcium/calmodulin-dependent serine proteinkinase (MAGUK family)), MAPT (microtubule-associated protein tau), SP1(Sp1 transcription factor), POLG (polymerase (DNA directed), gamma),AFF2 (AF4/FMR2 family, member 2), THBS1 (thrombospondin 1), TP53 (tumorprotein p53), ESR1 (estrogen receptor 1), CGGBP1 (CGG triplet repeatbinding protein 1), ABT1 (activator of basal transcription 1), KLK3(kallikrein-related peptidase 3), PRNP (prion protein), JUN (junoncogene), KCNN3 (potassium intermediate/small conductancecalcium-activated channel, subfamily N, member 3), BAX (BCL2-associatedX protein), FRAXA (fragile site, folic acid type, rare, fra(X)(q27.3) A(macroorchidism, mental retardation)), KBTBD10 (kelch repeat and BTB(POZ) domain containing 10), MBNL1 (muscleblind-like (Drosophila)),RAD51 (RAD51 homolog (RecA homolog, E. coli) (S. cerevisiae)), NCOA3(nuclear receptor coactivator 3), ERDA1 (expanded repeat domain, CAG/CTG1), TSC1 (tuberous sclerosis 1), COMP (cartilage oligomeric matrixprotein), GCLC (glutamate-cysteine ligase, catalytic subunit), RRAD(Ras-related associated with diabetes), MSH3 (mutS homolog 3 (E. coli)),DRD2 (dopamine receptor D2), CD44 (CD44 molecule (Indian blood group)),CTCF (CCCTC-binding factor (zinc finger protein)), CCND1 (cyclin D1),CLSPN (claspin homolog (Xenopus laevis)), MEF2A (myocyte enhancer factor2A), PTPRU (protein tyrosine phosphatase, receptor type, U), GAPDH(glyceraldehyde-3-phosphate dehydrogenase), TRIM22 (tripartitemotif-containing 22), WT1 (Wilms tumor 1), AHR (aryl hydrocarbonreceptor), GPX1 (glutathione peroxidase 1), TPMT (thiopurineS-methyltransferase), NDP (Norrie disease (pseudoglioma)), ARX(aristaless related homeobox), MUS81 (MUS81 endonuclease homolog (S.cerevisiae)), TYR (tyrosinase (oculocutaneous albinism 1A)), EGR1 (earlygrowth response 1), UNG (uracil-DNA glycosylase), NUMBL (numb homolog(Drosophila)-like), FABP2 (fatty acid binding protein 2, intestinal),EN2 (engrailed homeobox 2), CRYGC (crystallin, gamma C), SRP14 (signalrecognition particle 14 kDa (homologous Alu RNA binding protein)), CRYGB(crystallin, gamma B), PDCD1 (programmed cell death 1), HOXAl (homeoboxA1), ATXN2L (ataxin 2-like), PMS2 (PMS2 postmeiotic segregationincreased 2 (S. cerevisiae)), GLA (galactosidase, alpha), CBL (Cas-Br-M(murine) ecotropic retroviral transforming sequence), FTH1 (ferritin,heavy polypeptide 1), IL12RB2 (interleukin 12 receptor, beta 2), OTX2(orthodenticle homeobox 2), HOXA5 (homeobox A5), POLG2 (polymerase (DNAdirected), gamma 2, accessory subunit), DLX2 (distal-less homeobox 2),SIRPA (signal-regulatory protein alpha), OTX1 (orthodenticle homeobox1), AHRR (aryl-hydrocarbon receptor repressor), MANF (mesencephalicastrocyte-derived neurotrophic factor), TMEM158 (transmembrane protein158 (gene/pseudogene)), and ENSG00000078687.

Preferred proteins associated with trinucleotide repeat expansiondisorders include HTT (Huntingtin), AR (androgen receptor), FXN(frataxin), Atxn3 (ataxin), Atxn1 (ataxin), Atxn2 (ataxin), Atxn7(ataxin), Atxn10 (ataxin), DMPK (dystrophia myotonica-protein kinase),Atn1 (atrophin 1), CBP (creb binding protein), VLDLR (very low densitylipoprotein receptor), and any combination thereof.

Treating Hearing Diseases

The present invention also contemplates delivering the CRISPR-Cas systemto one or both ears.

Researchers are looking into whether gene therapy could be used to aidcurrent deafness treatments—namely, cochlear implants. Deafness is oftencaused by lost or damaged hair cells that cannot relay signals toauditory neurons. In such cases, cochlear implants may be used torespond to sound and transmit electrical signals to the nerve cells. Butthese neurons often degenerate and retract from the cochlea as fewergrowth factors are released by impaired hair cells.

US patent application 20120328580 describes injection of apharmaceutical composition into the ear (e.g., auricularadministration), such as into the luminae of the cochlea (e.g., theScala media, Sc vestibulae, and Sc tympani), e.g., using a syringe,e.g., a single-dose syringe. For example, one or more of the compoundsdescribed herein can be administered by intratympanic injection (e.g.,into the middle ear), and/or injections into the outer, middle, and/orinner ear. Such methods are routinely used in the art, for example, forthe administration of steroids and antibiotics into human ears.Injection can be, for example, through the round window of the ear orthrough the cochlear capsule. Other inner ear administration methods areknown in the art (see, e.g., Salt and Plontke, Drug Discovery Today,10:1299-1306, 2005).

In another mode of administration, the pharmaceutical composition can beadministered in situ, via a catheter or pump. A catheter or pump can,for example, direct a pharmaceutical composition into the cochlearluminae or the round window of the ear and/or the lumen of the colon.Exemplary drug delivery apparatus and methods suitable for administeringone or more of the compounds described herein into an ear, e.g., a humanear, are described by McKenna et al., (U.S. Publication No.2006/0030837) and Jacobsen et al., (U.S. Pat. No. 7,206,639). In someembodiments, a catheter or pump can be positioned, e.g., in the ear(e.g., the outer, middle, and/or inner ear) of a patient during asurgical procedure. In some embodiments, a catheter or pump can bepositioned, e.g., in the ear (e.g., the outer, middle, and/or inner ear)of a patient without the need for a surgical procedure.

Alternatively or in addition, one or more of the compounds describedherein can be administered in combination with a mechanical device suchas a cochlear implant or a hearing aid, which is worn in the outer ear.An exemplary cochlear implant that is suitable for use with the presentinvention is described by Edge et al., (U.S. Publication No.2007/0093878).

In some embodiments, the modes of administration described above may becombined in any order and can be simultaneous or interspersed.

Alternatively or in addition, the present invention may be administeredaccording to any of the Food and Drug Administration approved methods,for example, as described in CDER Data Standards Manual, version number004 (which is available at fda.give/cder/dsm/DRG/drg00301.htm).

In general, the cell therapy methods described in US patent application20120328580 can be used to promote complete or partial differentiationof a cell to or towards a mature cell type of the inner ear (e.g., ahair cell) in vitro. Cells resulting from such methods can then betransplanted or implanted into a patient in need of such treatment. Thecell culture methods required to practice these methods, includingmethods for identifying and selecting suitable cell types, methods forpromoting complete or partial differentiation of selected cells, methodsfor identifying complete or partially differentiated cell types, andmethods for implanting complete or partially differentiated cells aredescribed below.

Cells suitable for use in the present invention include, but are notlimited to, cells that are capable of differentiating completely orpartially into a mature cell of the inner ear, e.g., a hair cell (e.g.,an inner and/or outer hair cell), when contacted, e.g., in vitro, withone or more of the compounds described herein. Exemplary cells that arecapable of differentiating into a hair cell include, but are not limitedto stem cells (e.g., inner ear stem cells, adult stem cells, bone marrowderived stem cells, embryonic stem cells, mesenchymal stem cells, skinstem cells, iPS cells, and fat derived stem cells), progenitor cells(e.g., inner ear progenitor cells), support cells (e.g., Deiters' cells,pillar cells, inner phalangeal cells, tectal cells and Hensen's cells),and/or germ cells. The use of stem cells for the replacement of innerear sensory cells is described in Li et al., (U.S. Publication No.2005/0287127) and Li et al., (U.S. patent Ser. No. 11/953,797). The useof bone marrow derived stem cells for the replacement of inner earsensory cells is described in Edge et al., PCT/US2007/084654. iPS cellsare described, e.g., at Takahashi et al., Cell, Volume 131, Issue 5,Pages 861-872 (2007); Takahashi and Yamanaka, Cell 126, 663-76 (2006);Okita et al., Nature 448, 260-262 (2007); Yu, J. et al., Science318(5858):1917-1920 (2007); Nakagawa et al., Nat. Biotechnol. 26:101-106(2008); and Zaehres and Scholer, Cell 131(5):834-835 (2007). Suchsuitable cells can be identified by analyzing (e.g., qualitatively orquantitatively) the presence of one or more tissue specific genes. Forexample, gene expression can be detected by detecting the proteinproduct of one or more tissue-specific genes. Protein detectiontechniques involve staining proteins (e.g., using cell extracts or wholecells) using antibodies against the appropriate antigen. In this case,the appropriate antigen is the protein product of the tissue-specificgene expression. Although, in principle, a first antibody (i.e., theantibody that binds the antigen) can be labeled, it is more common (andimproves the visualization) to use a second antibody directed againstthe first (e.g., an anti-IgG). This second antibody is conjugated eitherwith fluorochromes, or appropriate enzymes for colorimetric reactions,or gold beads (for electron microscopy), or with the biotin-avidinsystem, so that the location of the primary antibody, and thus theantigen, can be recognized.

The CRISPR Cas molecules of the present invention may be delivered tothe ear by direct application of pharmaceutical composition to the outerear, with compositions modified from US Published application,20110142917. In some embodiments the pharmaceutical composition isapplied to the ear canal. Delivery to the ear may also be referred to asaural or optic delivery.

In some embodiments the RNA molecules of the invention are delivered inliposome or lipofectin formulations and the like and can be prepared bymethods well known to those skilled in the art. Such methods aredescribed, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and5,580,859, which are herein incorporated by reference.

Delivery systems aimed specifically at the enhanced and improveddelivery of siRNA into mammalian cells have been developed, (see, forexample, Shen et al FEBS Let. 2003, 539:111-114; Xia et al., Nat.Biotech. 2002, 20:1006-1010; Reich et al., Mol. Vision. 2003, 9:210-216; Sorensen et al., J. Mol. Biol. 2003, 327: 761-766; Lewis etal., Nat. Gen. 2002, 32: 107-108 and Simeoni et al., NAR 2003, 31, 11:2717-2724) and may be applied to the present invention. siRNA hasrecently been successfully used for inhibition of gene expression inprimates (see for example. Tolentino et al., Retina 24(4):660 which mayalso be applied to the present invention.

Qi et al. discloses methods for efficient siRNA transfection to theinner ear through the intact round window by a novel protein deliverytechnology which may be applied to the nucleic acid-targeting system ofthe present invention (see, e.g., Qi et al., Gene Therapy (2013), 1-9).In particular, a TAT double stranded RNA-binding domains (TAT-DRBDs),which can transfect Cy3-labeled siRNA into cells of the inner ear,including the inner and outer hair cells, crista ampullaris, maculautriculi and macula sacculi, through intact round-window permeation wassuccessful for delivering double stranded siRNAs in vivo for treatingvarious inner ear ailments and preservation of hearing function. About40 μl of 10 mM RNA may be contemplated as the dosage for administrationto the ear.

According to Rejali et al. (Hear Res. 2007 June; 228(1-2):180-7),cochlear implant function can be improved by good preservation of thespiral ganglion neurons, which are the target of electrical stimulationby the implant and brain derived neurotrophic factor (BDNF) haspreviously been shown to enhance spiral ganglion survival inexperimentally deafened ears. Rejali et al. tested a modified design ofthe cochlear implant electrode that includes a coating of fibroblastcells transduced by a viral vector with a BDNF gene insert. Toaccomplish this type of ex vivo gene transfer, Rejali et al. transducedguinea pig fibroblasts with an adenovirus with a BDNF gene cassetteinsert, and determined that these cells secreted BDNF and then attachedBDNF-secreting cells to the cochlear implant electrode via an agarosegel, and implanted the electrode in the scala tympani. Rejali et al.determined that the BDNF expressing electrodes were able to preservesignificantly more spiral ganglion neurons in the basal turns of thecochlea after 48 days of implantation when compared to controlelectrodes and demonstrated the feasibility of combining cochlearimplant therapy with ex vivo gene transfer for enhancing spiral ganglionneuron survival. Such a system may be applied to the nucleicacid-targeting system of the present invention for delivery to the ear.

Mukherjea et al. (Antioxidants & Redox Signaling, Volume 13, Number 5,2010) document that knockdown of NOX3 using short interfering (si) RNAabrogated cisplatin ototoxicity, as evidenced by protection of OHCs fromdamage and reduced threshold shifts in auditory brainstem responses(ABRs). Different doses of siNOX3 (0.3, 0.6, and 0.9 μg) wereadministered to rats and NOX3 expression was evaluated by real timeRT-PCR. The lowest dose of NOX3 siRNA used (0.3 μg) did not show anyinhibition of NOX3 mRNA when compared to transtympanic administration ofscrambled siRNA or untreated cochleae. However, administration of thehigher doses of NOX3 siRNA (0.6 and 0.9 μg) reduced NOX3 expressioncompared to control scrambled siRNA. Such a system may be applied to theCRISPR Cas system of the present invention for transtympanicadministration with a dosage of about 2 mg to about 4 mg of CRISPR Casfor administration to a human.

Jung et al. (Molecular Therapy, vol. 21 no. 4, 834-841 April 2013)demonstrate that Hes5 levels in the utricle decreased after theapplication of siRNA and that the number of hair cells in these utricleswas significantly larger than following control treatment. The datasuggest that siRNA technology may be useful for inducing repair andregeneration in the inner ear and that the Notch signaling pathway is apotentially useful target for specific gene expression inhibition. Junget al. injected 8 μg of Hes5 siRNA in 2 μl volume, prepared by addingsterile normal saline to the lyophilized siRNA to a vestibularepithelium of the ear. Such a system may be applied to the nucleicacid-targeting system of the present invention for administration to thevestibular epithelium of the ear with a dosage of about 1 to about 30 mgof CRISPR Cas for administration to a human.

Gene Targeting in Non-Dividing Cells (Neurons & Muscle)

Non-dividing (especially non-dividing, fully differentiated) cell typespresent issues for gene targeting or genome engineering, for examplebecause homologous recombination (HR) is generally suppressed in the G1cell-cycle phase. However, while studying the mechanisms by which cellscontrol normal DNA repair systems, Durocher discovered a previouslyunknown switch that keeps HR “off′ in non-dividing cells and devised astrategy to toggle this switch back on. Orthwein et al. (DanielDurocher's lab at the Mount Sinai Hospital in Ottawa, Canada) recentlyreported (Nature 16142, published online 9 Dec. 2015) have shown thatthe suppression of HR can be lifted and gene targeting successfullyconcluded in both kidney (293T) and osteosarcoma (U20S) cells. Tumorsuppressors, BRCA1, PALB2 and BRAC2 are known to promote DNA DSB repairby HR. They found that formation of a complex of BRCA1 with PALB2-BRAC2is governed by a ubiquitin site on PALB2, such that action on the siteby an E3 ubiquitin ligase. This E3 ubiquitin ligase is composed of KEAP1(a PALB2-interacting protein) in complex with cullin-3 (CUL3)-RBX1.PALB2 ubiquitylation suppresses its interaction with BRCA1 and iscounteracted by the deubiquitylase USP11, which is itself under cellcycle control. Restoration of the BRCA1-PALB2 interaction combined withthe activation of DNA-end resection is sufficient to induce homologousrecombination in G1, as measured by a number of methods including aCRISPR-Cas9-based gene-targeting assay directed at USP11 or KEAP1(expressed from a pX459 vector). However, when the BRCA1-PALB2interaction was restored in resection-competent G1 cells using eitherKEAP1 depletion or expression of the PALB2-KR mutant, a robust increasein gene-targeting events was detected.

Thus, reactivation of HR in cells, especially non-dividing, fullydifferentiated cell types is preferred, in some embodiments. In someembodiments, promotion of the BRCA1-PALB2 interaction is preferred insome embodiments. In some embodiments, the target ell is a non-dividingcell. In some embodiments, the target cell is a neurone or muscle cell.In some embodiments, the target cell is targeted in vivo. In someembodiments, the cell is in G1 and HR is supressed. In some embodiments,use of KEAP1 depletion, for example inhibition of expression of KEAP1activity, is preferred. KEAP1 depletion may be achieved through siRNA,for example as shown in Orthwein et al. Alternatively, expression of thePALB2-KR mutant (lacking all eight Lys residues in the BRCA1-interactiondomain is preferred, either in combination with KEAP1 depletion oralone. PALB2-KR interacts with BRCA1 irrespective of cell cycleposition. Thus, promotion or restoration of the BRCA1-PALB2 interaction,especially in G1 cells, is preferred in some embodiments, especiallywhere the target cells are non-dividing, or where removal and return (exvivo gene targeting) is problematic, for example neurone or musclecells. KEAP1 siRNA is available from Thermo Fisher. In some embodiments,a BRCA1-PALB2 complex may be delivered to the G1 cell. In someembodiments, PALB2 deubiquitylation may be promoted for example byincreased expression of the deubiquitylase USP11, so it is envisagedthat a construct may be provided to promote or up-regulate expression oractivity of the deubiquitylase USP11.

Treating Diseases of the Eve

The present invention also contemplates delivering the CRISPR-Cas systemto one or both eyes.

In yet another aspect of the invention, the CRISPR-Cas system may beused to correct ocular defects that arise from several genetic mutationsfurther described in Genetic Diseases of the Eye, Second Edition, editedby Elias I. Traboulsi, Oxford University Press, 2012.

For administration to the eye, lentiviral vectors, in particular equineinfectious anemia viruses (EIAV) are particularly preferred.

In another embodiment, minimal non-primate lentiviral vectors based onthe equine infectious anemia virus (EIAV) are also contemplated,especially for ocular gene therapy (see, e.g., Balagaan, J Gene Med2006; 8: 275-285, Published online 21 Nov. 2005 in Wiley InterScience(www.interscience.wiley.com). DOI: 10.1002/jgm.845). The vectors arecontemplated to have cytomegalovirus (CMV) promoter driving expressionof the target gene. Intracameral, subretinal, intraocular andintravitreal injections are all contemplated (see, e.g., Balagaan, JGene Med 2006; 8: 275-285, Published online 21 Nov. 2005 in WileyInterScience (www.interscience.wiley.com). DOI: 10.1002/jgm.845).Intraocular injections may be performed with the aid of an operatingmicroscope. For subretinal and intravitreal injections, eyes may beprolapsed by gentle digital pressure and fundi visualised using acontact lens system consisting of a drop of a coupling medium solutionon the cornea covered with a glass microscope slide coverslip. Forsubretinal injections, the tip of a 10-mm 34-gauge needle, mounted on a5-μl Hamilton syringe may be advanced under direct visualisation throughthe superior equatorial sclera tangentially towards the posterior poleuntil the aperture of the needle was visible in the subretinal space.Then, 2 μl of vector suspension may be injected to produce a superiorbullous retinal detachment, thus confirming subretinal vectoradministration. This approach creates a self-sealing sclerotomy allowingthe vector suspension to be retained in the subretinal space until it isabsorbed by the RPE, usually within 48 h of the procedure. Thisprocedure may be repeated in the inferior hemisphere to produce aninferior retinal detachment. This technique results in the exposure ofapproximately 70% of neurosensory retina and RPE to the vectorsuspension. For intravitreal injections, the needle tip may be advancedthrough the sclera 1 mm posterior to the corneoscleral limbus and 2 μlof vector suspension injected into the vitreous cavity. For intracameralinjections, the needle tip may be advanced through a corneosclerallimbal paracentesis, directed towards the central cornea, and 2 μl ofvector suspension may be injected. For intracameral injections, theneedle tip may be advanced through a corneoscleral limbal paracentesis,directed towards the central cornea, and 2 μl of vector suspension maybe injected. These vectors may be injected at titres of either1.0-1.4×10¹⁰ or 1.0-1.4×10¹ transducing units (TU)/ml.

In another embodiment, RetinoStat®, an equine infectious anemiavirus-based lentiviral gene therapy vector that expresses angiostaticproteins endostatin and angiostatin that is delivered via a subretinalinjection for the treatment of the web form of age-related maculardegeneration is also contemplated (see, e.g., Binley et al., HUMAN GENETHERAPY 23:980-991 (September 2012)). Such a vector may be modified forthe CRISPR-Cas system of the present invention. Each eye may be treatedwith either RetinoStat® at a dose of 1.1×10¹ transducing units per eye(TU/eye) in a total volume of 100 μl.

In another embodiment, an E1-, partial E3-, E4-deleted adenoviral vectormay be contemplated for delivery to the eye. Twenty-eight patients withadvanced neovascular agerelated macular degeneration (AMD) were given asingle intravitreous injection of an E1-, partial E3-, E4-deletedadenoviral vector expressing human pigment ep-ithelium-derived factor(AdPEDF.ll) (see, e.g., Campochiaro et al., Human Gene Therapy17:167-176 (February 2006)). Doses ranging from 10⁶ to 10^(9.5) particleunits (PU) were investigated and there were no serious adverse eventsrelated to AdPEDF.ll and no dose-limiting toxicities (see, e.g.,Campochiaro et al., Human Gene Therapy 17:167-176 (February 2006)).Adenoviral vectormediated ocular gene transfer appears to be a viableapproach for the treatment of ocular disorders and could be applied tothe CRISPR Cas system.

In another embodiment, the sd-rxRNA® system of RXi Pharmaceuticals maybe used and/or adapted for delivering CRISPR Cas to the eye. In thissystem, a single intravitreal administration of 3 μg of sd-rxRNA resultsin sequence-specific reduction of PPIB mRNA levels for 14 days. Thesd-rxRNA® system may be applied to the nucleic acid-targeting system ofthe present invention, contemplating a dose of about 3 to 20 mg ofCRISPR administered to a human.

Millington-Ward et al. (Molecular Therapy, vol. 19 no. 4, 642-649 April2011) describes adeno-associated virus (AAV) vectors to deliver an RNAinterference (RNAi)-based rhodopsin suppressor and a codon-modifiedrhodopsin replacement gene resistant to suppression due to nucleotidealterations at degenerate positions over the RNAi target site. Aninjection of either 6.0×10⁸ vp or 1.8×10¹⁰ vp AAV were subretinallyinjected into the eyes by Millington-Ward et al. The AAV vectors ofMillington-Ward et al. may be applied to the CRISPR Cas system of thepresent invention, contemplating a dose of about 2×10¹¹ to about 6×10¹¹vp administered to a human.

Dalkara et al. (Sci Transl Med 5, 189ra76 (2013)) also relates to invivo directed evolution to fashion an AAV vector that delivers wild-typeversions of defective genes throughout the retina after noninjuriousinjection into the eyes' vitreous humor. Dalkara describes a 7merpeptide display library and an AAV library constructed by DNA shufflingof cap genes from AAV1, 2, 4, 5, 6, 8, and 9. The rcAAV libraries andrAAV vectors expressing GFP under a CAG or Rho promoter were packagedand deoxyribonuclease-resistant genomic titers were obtained throughquantitative PCR. The libraries were pooled, and two rounds of evolutionwere performed, each consisting of initial library diversificationfollowed by three in vivo selection steps. In each such step, P30rho-GFP mice were intravitreally injected with 2 ml ofiodixanol-purified, phosphate-buffered saline (PBS)-dialyzed librarywith a genomic titer of about 1×10¹² vg/ml. The AAV vectors of Dalkaraet al. may be applied to the nucleic acid-targeting system of thepresent invention, contemplating a dose of about 1×10¹⁵ to about 1×10¹⁶vg/ml administered to a human.

In another embodiment, the rhodopsin gene may be targeted for thetreatment of retinitis pigmentosa (RP), wherein the system of US PatentPublication No. 20120204282 assigned to Sangamo BioSciences, Inc. may bemodified in accordance of the CRISPR Cas system of the presentinvention.

In another embodiment, the methods of US Patent Publication No.20130183282 assigned to Cellectis, which is directed to methods ofcleaving a target sequence from the human rhodopsin gene, may also bemodified to the nucleic acid-targeting system of the present invention.

US Patent Publication No. 20130202678 assigned to Academia Sinicarelates to methods for treating retinopathies and sight-threateningophthalmologic disorders relating to delivering of the Puf-A gene (whichis expressed in retinal ganglion and pigmented cells of eye tissues anddisplays a unique anti-apoptotic activity) to the sub-retinal orintravitreal space in the eye. In particular, desirable targets arezgc:193933, prdm1a, spata2, tex10, rbb4, ddx3, zp2.2, Blimp-1 and HtrA2,all of which may be targeted by the nucleic acid-targeting system of thepresent invention.

Wu (Cell Stem Cell, 13:659-62, 2013) designed a guide RNA that led Cas9to a single base pair mutation that causes cataracts in mice, where itinduced DNA cleavage. Then using either the other wild-type allele oroligos given to the zygotes repair mechanisms corrected the sequence ofthe broken allele and corrected the cataract-causing genetic defect inmutant mouse.

US Patent Publication No. 20120159653, describes use of zinc fingernucleases to genetically modify cells, animals and proteins associatedwith macular degeneration (MD). Macular degeneration (MD) is the primarycause of visual impairment in the elderly, but is also a hallmarksymptom of childhood diseases such as Stargardt disease, Sorsby fundus,and fatal childhood neurodegenerative diseases, with an age of onset asyoung as infancy. Macular degeneration results in a loss of vision inthe center of the visual field (the macula) because of damage to theretina. Currently existing animal models do not recapitulate majorhallmarks of the disease as it is observed in humans. The availableanimal models comprising mutant genes encoding proteins associated withMD also produce highly variable phenotypes, making translations to humandisease and therapy development problematic.

One aspect of US Patent Publication No. 20120159653 relates to editingof any chromosomal sequences that encode proteins associated with MDwhich may be applied to the nucleic acid-targeting system of the presentinvention. The proteins associated with MD are typically selected basedon an experimental association of the protein associated with MD to anMD disorder. For example, the production rate or circulatingconcentration of a protein associated with MD may be elevated ordepressed in a population having an MD disorder relative to a populationlacking the MD disorder. Differences in protein levels may be assessedusing proteomic techniques including but not limited to Western blot,immunohistochemical staining, enzyme linked immunosorbent assay (ELISA),and mass spectrometry. Alternatively, the proteins associated with MDmay be identified by obtaining gene expression profiles of the genesencoding the proteins using genomic techniques including but not limitedto DNA microarray analysis, serial analysis of gene expression (SAGE),and quantitative real-time polymerase chain reaction (Q-PCR).

By way of non-limiting example, proteins associated with MD include butare not limited to the following proteins: (ABCA4) ATP-binding cassette,sub-family A (ABC1), member 4 ACHM1 achromatopsia (rod monochromacy) 1ApoE Apolipoprotein E (ApoE) CIQTNF5 (CTRP5) C1q and tumor necrosisfactor related protein 5 (CIQTNF5) C2 Complement component 2 (C2) C3Complement components (C3) CCL2 Chemokine (C—C motif) Ligand 2 (CCL2)CCR2 Chemokine (C—C motif) receptor 2 (CCR2) CD36 Cluster ofDifferentiation 36 CFB Complement factor B CFH Complement factor CFH HCFHR1 complement factor H-related 1 CFHR3 complement factor H-related 3CNGB3 cyclic nucleotide gated channel beta 3 CP ceruloplasmin (CP) CRP Creactive protein (CRP) CST3 cystatin C or cystatin 3 (CST3) CTSDCathepsin D (CTSD) CX3CR1 chemokine (C-X3-C motif) receptor 1 ELOVL4Elongation of very long chain fatty acids 4 ERCC6 excision repaircrosscomplementing rodent repair deficiency, complementation group 6FBLN5 Fibulin-5 FBLN5 Fibulin 5 FBLN6 Fibulin 6 FSCN2 fascin (FSCN2)HMCN1 Hemicentin 1 HMCN1 hemicentin 1 HTRA1 HtrA serine peptidase 1(HTRA1) HTRA1 HtrA serine peptidase 1 IL-6 Interleukin 6 IL-8Interleukin 8 LOC387715 Hypothetical protein PLEKHA1 Pleckstrin homologydomaincontaining family A member 1 (PLEKHA1) PROM1 Prominin 1(PROM1 orCD133) PRPH2 Peripherin-2 RPGR retinitis pigmentosa GTPase regulatorSERPINGI serpin peptidase inhibitor, clade G, member 1 (C1-inhibitor)TCOF1 Treacle TIMP3 Metalloproteinase inhibitor 3 (TIMP3) TLR3 Toll-likereceptor 3.

The identity of the protein associated with MD whose chromosomalsequence is edited can and will vary. In preferred embodiments, theproteins associated with MD whose chromosomal sequence is edited may bethe ATP-binding cassette, sub-family A (ABC1) member 4 protein (ABCA4)encoded by the ABCR gene, the apolipoprotein E protein (APOE) encoded bythe APOE gene, the chemokine (C—C motif) Ligand 2 protein (CCL2) encodedby the CCL2 gene, the chemokine (C—C motif) receptor 2 protein (CCR2)encoded by the CCR2 gene, the ceruloplasmin protein (CP) encoded by theCP gene, the cathepsin D protein (CTSD) encoded by the CTSD gene, or themetalloproteinase inhibitor 3 protein (TIMP3) encoded by the TIMP3 gene.In an exemplary embodiment, the genetically modified animal is a rat,and the edited chromosomal sequence encoding the protein associated withMD may be: (ABCA4) ATPbinding cassette, NM_000350 sub-family A (ABC1),member 4 APOE Apolipoprotein E NM_138828 (APOE) CCL2 Chemokine (C—CNM_031530 motif) Ligand 2 (CCL2) CCR2 Chemokine (C—C NM_021866 motif)receptor 2 (CCR2) CP ceruloplasmin (CP) NM_012532 CTSD Cathepsin D(CTSD) NM_134334 TIMP3 Metalloproteinase NM_012886 inhibitor 3 (TIMP3)The animal or cell may comprise 1, 2, 3, 4, 5, 6, 7 or more disruptedchromosomal sequences encoding a protein associated with MD and zero, 1,2, 3, 4, 5, 6, 7 or more chromosomally integrated sequences encoding thedisrupted protein associated with MD.

The edited or integrated chromosomal sequence may be modified to encodean altered protein associated with MD. Several mutations in MD-relatedchromosomal sequences have been associated with MD. Non-limitingexamples of mutations in chromosomal sequences associated with MDinclude those that may cause MD including in the ABCR protein, E471K(i.e. glutamate at position 471 is changed to lysine), RI129L (i.e.arginine at position 1129 is changed to leucine), T1428M (i.e. threonineat position 1428 is changed to methionine), R1517S (i.e. arginine atposition 1517 is changed to serine), I1562T (i.e. isoleucine at position1562 is changed to threonine), and G1578R (i.e. glycine at position 1578is changed to arginine); in the CCR2 protein, V64I (i.e. valine atposition 192 is changed to isoleucine); in CP protein, G969B (i.e.glycine at position 969 is changed to asparagine or aspartate); in TIMP3protein, S156C (i.e. serine at position 156 is changed to cysteine),G166C (i.e. glycine at position 166 is changed to cysteine), G167C (i.e.glycine at position 167 is changed to cysteine), Y168C (i.e. tyrosine atposition 168 is changed to cysteine), S170C (i.e. serine at position 170is changed to cysteine), Y172C (i.e. tyrosine at position 172 is changedto cysteine) and S181C (i.e. serine at position 181 is changed tocysteine). Other associations of genetic variants in MD-associated genesand disease are known in the art.

Treating Circulatory and Muscular Diseases

The present invention also contemplates delivering the CRISPR-Cas systemdescribed herein, e.g. C2c1 or C2c3 effector protein systems, to theheart. For the heart, a myocardium tropic adena-associated virus (AAVM)is preferred, in particular AAVM41 which showed preferential genetransfer in the heart (see, e.g., Lin-Yanga et al., PNAS, Mar. 10, 2009,vol. 106, no. 10). Administration may be systemic or local. A dosage ofabout 1-10×10¹⁴ vector genomes are contemplated for systemicadministration. See also, e.g., Eulalio et al. (2012) Nature 492: 376and Somasuntharam et al. (2013) Biomaterials 34: 7790.

For example, US Patent Publication No. 20110023139, describes use ofzinc finger nucleases to genetically modify cells, animals and proteinsassociated with cardiovascular disease. Cardiovascular diseasesgenerally include high blood pressure, heart attacks, heart failure, andstroke and TIA. Any chromosomal sequence involved in cardiovasculardisease or the protein encoded by any chromosomal sequence involved incardiovascular disease may be utilized in the methods described in thisdisclosure. The cardiovascular-related proteins are typically selectedbased on an experimental association of the cardiovascular-relatedprotein to the development of cardiovascular disease. For example, theproduction rate or circulating concentration of a cardiovascular-relatedprotein may be elevated or depressed in a population having acardiovascular disorder relative to a population lacking thecardiovascular disorder. Differences in protein levels may be assessedusing proteomic techniques including but not limited to Western blot,immunohistochemical staining, enzyme linked immunosorbent assay (ELISA),and mass spectrometry. Alternatively, the cardiovascular-relatedproteins may be identified by obtaining gene expression profiles of thegenes encoding the proteins using genomic techniques including but notlimited to DNA microarray analysis, serial analysis of gene expression(SAGE), and quantitative real-time polymerase chain reaction (Q-PCR).

By way of example, the chromosomal sequence may comprise, but is notlimited to, ILIB (interleukin 1, beta), XDH (xanthine dehydrogenase),TP53 (tumor protein p53), PTGIS (prostaglandin 12 (prostacyclin)synthase), MB (myoglobin), I14 (interleukin 4), ANGPT1 (angiopoietin 1),ABCG8 (ATP-binding cassette, sub-family G (WHITE), member 8), CTSK(cathepsin K), PTGIR (prostaglandin 12 (prostacyclin) receptor (IP)),KCNJ11 (potassium inwardly-rectifying channel, subfamily J, member 11),INS (insulin), CRP (C-reactive protein, pentraxin-related), PDGFRB(platelet-derived growth factor receptor, beta polypeptide), CCNA2(cyclin A2), PDGFB (platelet-derived growth factor beta polypeptide(simian sarcoma viral (v-sis) oncogene homolog)), KCNJ5 (potassiuminwardly-rectifying channel, subfamily J, member 5), KCNN3 (potassiumintermediate/small conductance calcium-activated channel, subfamily N,member 3), CAPN10 (calpain 10), PTGES (prostaglandin E synthase), ADRA2B(adrenergic, alpha-2B-, receptor), ABCG5 (ATP-binding cassette,sub-family G (WHITE), member 5), PRDX2 (peroxiredoxin 2), CAPN5 (calpain5), PARP14 (poly (ADP-ribose) polymerase family, member 14), MEX3C(mex-3 homolog C (C. elegans)), ACE angiotensin I converting enzyme(peptidyl-dipeptidase A) 1), TNF (tumor necrosis factor (TNFsuperfamily, member 2)), IL6 (interleukin 6 (interferon, beta 2)), STN(statin), SERPINE1 (serpin peptidase inhibitor, clade E (nexin,plasminogen activator inhibitor type 1), member 1), ALB (albumin),ADIPOQ (adiponectin, C1Q and collagen domain containing), APOB(apolipoprotein B (including Ag(x) antigen)), APOE (apolipoprotein E),LEP (leptin), MTHFR (5,10-methylenetetrahydrofolate reductase (NADPH)),APOA1 (apolipoprotein A-I), EDN1 (endothelin 1), NPPB (natriureticpeptide precursor B), NOS3 (nitric oxide synthase 3 (endothelial cell)),PPARG (peroxisome proliferator-activated receptor gamma), PLAT(plasminogen activator, tissue), PTGS2 (prostaglandin-endoperoxidesynthase 2 (prostaglandin G/H synthase and cyclooxygenase)), CETP(cholesteryl ester transfer protein, plasma), AGTR1 (angiotensin IIreceptor, type 1), HMGCR (3-hydroxy-3-methylglutaryl-Coenzyme Areductase), IGF1 (insulin-like growth factor 1 (somatomedin C)), SELE(selectin E), REN (renin), PPARA (peroxisome proliferator-activatedreceptor alpha), PON1 (paraoxonase 1), KNG1 (kininogen 1), CCL2(chemokine (C—C motif) ligand 2), LPL (lipoprotein lipase), VWF (vonWillebrand factor), F2 (coagulation factor II (thrombin)), ICAM1(intercellular adhesion molecule 1), TGFB1 (transforming growth factor,beta 1), NPPA (natriuretic peptide precursor A), I110 (interleukin 10),EPO (erythropoietin), SOD1 (superoxide dismutase 1, soluble), VCAM1(vascular cell adhesion molecule 1), IFNG (interferon, gamma), LPA(lipoprotein, Lp(a)), MPO (myeloperoxidase), ESR1 (estrogen receptor 1),MAPK1 (mitogen-activated protein kinase 1), HP (haptoglobin), F3(coagulation factor III (thromboplastin, tissue factor)), CST3 (cystatinC), COG2 (component of oligomeric golgi complex 2), MMP9 (matrixmetallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IVcollagenase)), SERPINC1 (serpin peptidase inhibitor, clade C(antithrombin), member 1), F8 (coagulation factor VIII, procoagulantcomponent), HMOX1 (heme oxygenase (decycling) 1), APOC3 (apolipoproteinC-III), IL8 (interleukin 8), PROK (prokineticin 1), CBS(cystathionine-beta-synthase), NOS2 (nitric oxide synthase 2,inducible), TLR4 (toll-like receptor 4), SELP (selectin P (granulemembrane protein 140 kDa, antigen CD62)), ABCA1 (ATP-binding cassette,sub-family A (ABC1), member 1), AGT (angiotensinogen (serpin peptidaseinhibitor, clade A, member 8)), LDLR (low density lipoprotein receptor),GPT (glutamic-pyruvate transaminase (alanine aminotransferase)), VEGFA(vascular endothelial growth factor A), NR3C2 (nuclear receptorsubfamily 3, group C, member 2), IL18 (interleukin 18(interferon-gamma-inducing factor)), NOS1 (nitric oxide synthase 1(neuronal)), NR3C1 (nuclear receptor subfamily 3, group C, member 1(glucocorticoid receptor)), FGB (fibrinogen beta chain), HGF (hepatocytegrowth factor (hepapoietin A; scatter factor)), ILIA (interleukin 1,alpha), RETN (resistin), AKT1 (v-akt murine thymoma viral oncogenehomolog 1), LIPC (lipase, hepatic), HSPD1 (heat shock 60 kDa protein 1(chaperonin)), MAPK14 (mitogen-activated protein kinase 14), SPP1(secreted phosphoprotein 1), ITGB3 (integrin, beta 3 (plateletglycoprotein llla, antigen CD61)), CAT (catalase), UTS2 (urotensin 2),THBD (thrombomodulin), F10 (coagulation factor X), CP (ceruloplasmin(ferroxidase)), TNFRSF11B (tumor necrosis factor receptor superfamily,member 11b), EDNRA (endothelin receptor type A), EGFR (epidermal growthfactor receptor (erythroblastic leukemia viral (v-erb-b) oncogenehomolog, avian)), MMP2 (matrix metallopeptidase 2 (gelatinase A, 72 kDagelatinase, 72 kDa type IV collagenase)), PLG (plasminogen), NPY(neuropeptide Y), RHOD (ras homolog gene family, member D), MAPK8(mitogen-activated protein kinase 8), MYC (v-myc myelocytomatosis viraloncogene homolog (avian)), FN1 (fibronectin 1), CMA1 (chymase 1, mastcell), PLAU (plasminogen activator, urokinase), GNB3 (guanine nucleotidebinding protein (G protein), beta polypeptide 3), ADRB2 (adrenergic,beta-2-, receptor, surface), APOA5 (apolipoprotein A-V), SOD2(superoxide dismutase 2, mitochondrial), F5 (coagulation factor V(proaccelerin, labile factor)), VDR (vitamin D (1,25-dihydroxyvitaminD3) receptor), ALOX5 (arachidonate 5-lipoxygenase), HLA-DRB1 (majorhistocompatibility complex, class II, DR beta 1), PARP1 (poly(ADP-ribose) polymerase 1), CD40LG (CD40 ligand), PON2 (paraoxonase 2),AGER (advanced glycosylation end product-specific receptor), IRS1(insulin receptor substrate 1), PTGS1 (prostaglandin-endoperoxidesynthase 1 (prostaglandin G/H synthase and cyclooxygenase)), ECE1(endothelin converting enzyme 1), F7 (coagulation factor VII (serumprothrombin conversion accelerator)), URN (interleukin 1 receptorantagonist), EPHX2 (epoxide hydrolase 2, cytoplasmic), IGFBP1(insulin-like growth factor binding protein 1), MAPK10(mitogen-activated protein kinase 10), FAS (Fas (TNF receptorsuperfamily, member 6)), ABCB1 (ATP-binding cassette, sub-family B(MDR/TAP), member 1), JUN (jun oncogene), IGFBP3 (insulin-like growthfactor binding protein 3), CD14 (CD14 molecule), PDE5A(phosphodiesterase 5A, cGMP-specific), AGTR2 (angiotensin II receptor,type 2), CD40 (CD40 molecule, TNF receptor superfamily member 5), LCAT(lecithin-cholesterol acyltransferase), CCR5 (chemokine (C—C motif)receptor 5), MMP1 (matrix metallopeptidase 1 (interstitialcollagenase)), TIMP1 (TIMP metallopeptidase inhibitor 1), ADM(adrenomedullin), DYT10 (dystonia 10), STAT3 (signal transducer andactivator of transcription 3 (acute-phase response factor)), MMP3(matrix metallopeptidase 3 (stromelysin 1, progelatinase)), ELN(elastin), USF1 (upstream transcription factor 1), CFH (complementfactor H), HSPA4 (heat shock 70 kDa protein 4), MMP12 (matrixmetallopeptidase 12 (macrophage elastase)), MME (membranemetallo-endopeptidase), F2R (coagulation factor II (thrombin) receptor),SELL (selectin L), CTSB (cathepsin B), ANXA5 (annexin A5), ADRB1(adrenergic, beta-I-, receptor), CYBA (cytochrome b-245, alphapolypeptide), FGA (fibrinogen alpha chain), GGT1(gamma-glutamyltransferase 1), LIPG (lipase, endothelial), HIFlA(hypoxia inducible factor 1, alpha subunit (basic helix-loop-helixtranscription factor)), CXCR4 (chemokine (C—X—C motif) receptor 4), PROC(protein C (inactivator of coagulation factors Va and VIIIa)), SCARB1(scavenger receptor class B, member 1), CD79A (CD79a molecule,immunoglobulin-associated alpha), PLTP (phospholipid transfer protein),ADD1 (adducin 1 (alpha)), FGG (fibrinogen gamma chain), SAA1 (serumamyloid A1), KCNH2 (potassium voltage-gated channel, subfamily H(eag-related), member 2), DPP4 (dipeptidyl-peptidase 4), G6PD(glucose-6-phosphate dehydrogenase), NPR1 (natriuretic peptide receptorA/guanylate cyclase A (atrionatriuretic peptide receptor A)), VTN(vitronectin), KIAA0101 (KIAA0101), FOS (FBJ murine osteosarcoma viraloncogene homolog), TLR2 (toll-like receptor 2), PPIG (peptidylprolylisomerase G (cyclophilin G)), ILIR1 (interleukin 1 receptor, type I), AR(androgen receptor), CYP1A1 (cytochrome P450, family 1, subfamily A,polypeptide 1), SERPINA1 (serpin peptidase inhibitor, clade A (alpha-1antiproteinase, antitrypsin), member 1), MTR(5-methyltetrahydrofolate-homocysteine methyltransferase), RBP4 (retinolbinding protein 4, plasma), APOA4 (apolipoprotein A-IV), CDKN2A(cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4)),FGF2 (fibroblast growth factor 2 (basic)), EDNRB (endothelin receptortype B), ITGA2 (integrin, alpha 2 (CD49B, alpha 2 subunit of VLA-2receptor)), CABIN1 (calcineurin binding protein 1), SHBG (sexhormone-binding globulin), HMGB1 (high-mobility group box 1), HSP90B2P(heat shock protein 90 kDa beta (Grp94), member 2 (pseudogene)), CYP3A4(cytochrome P450, family 3, subfamily A, polypeptide 4), GJA1 (gapjunction protein, alpha 1, 43 kDa), CAV1 (caveolin 1, caveolae protein,22 kDa), ESR2 (estrogen receptor 2 (ER beta)), LTA (lymphotoxin alpha(TNF superfamily, member 1)), GDF15 (growth differentiation factor 15),BDNF (brain-derived neurotrophic factor), CYP2D6 (cytochrome P450,family 2, subfamily D, polypeptide 6), NGF (nerve growth factor (betapolypeptide)), SP1 (Sp1 transcription factor), TGIF1 (TGFB-inducedfactor homeobox 1), SRC (v-src sarcoma (Schmidt-Ruppin A-2) viraloncogene homolog (avian)), EGF (epidermal growth factor(beta-urogastrone)), PIK3CG (phosphoinositide-3-kinase, catalytic, gammapolypeptide), HLA-A (major histocompatibility complex, class I, A),KCNQ1 (potassium voltage-gated channel, KQT-like subfamily, member 1),CNR1 (cannabinoid receptor 1 (brain)), FBN1 (fibrillin 1), CHKA (cholinekinase alpha), BEST1 (bestrophin 1), APP (amyloid beta (A4) precursorprotein), CTNNB1 (catenin (cadherin-associated protein), beta 1, 88kDa), IL2 (interleukin 2), CD36 (CD36 molecule (thrombospondinreceptor)), PRKAB1 (protein kinase, AMP-activated, beta 1 non-catalyticsubunit), TPO (thyroid peroxidase), ALDH7A1 (aldehyde dehydrogenase 7family, member A1), CX3CR1 (chemokine (C-X3-C motif) receptor 1), TH(tyrosine hydroxylase), F9 (coagulation factor IX), GH1 (growth hormone1), TF (transferrin), HFE (hemochromatosis), IL17A (interleukin 17A),PTEN (phosphatase and tensin homolog), GSTM1 (glutathione S-transferasemu 1), DMD (dystrophin), GATA4 (GATA binding protein 4), F13A1(coagulation factor XIII, A1 polypeptide), TTR (transthyretin), FABP4(fatty acid binding protein 4, adipocyte), PON3 (paraoxonase 3), APOC1(apolipoprotein C-I), INSR (insulin receptor), TNFRSF1B (tumor necrosisfactor receptor superfamily, member 1B), HTR2A (5-hydroxytryptamine(serotonin) receptor 2A), CSF3 (colony stimulating factor 3(granulocyte)), CYP2C9 (cytochrome P450, family 2, subfamily C,polypeptide 9), TXN (thioredoxin), CYP11B2 (cytochrome P450, family 11,subfamily B, polypeptide 2), PTH (parathyroid hormone), CSF2 (colonystimulating factor 2 (granulocyte-macrophage)), KDR (kinase insertdomain receptor (a type III receptor tyrosine kinase)), PLA2G2A(phospholipase A2, group IIA (platelets, synovial fluid)), B2M(beta-2-microglobulin), THBS1 (thrombospondin 1), GCG (glucagon), RHOA(ras homolog gene family, member A), ALDH2 (aldehyde dehydrogenase 2family (mitochondrial)), TCF7L2 (transcription factor 7-like 2 (T-cellspecific, HMG-box)), BDKRB2 (bradykinin receptor B2), NFE2L2 (nuclearfactor (erythroid-derived 2)-like 2), NOTCH1 (Notch homolog 1,translocation-associated (Drosophila)), UGT1A1 (UDPglucuronosyltransferase 1 family, polypeptide A1), IFNA1 (interferon,alpha 1), PPARD (peroxisome proliferator-activated receptor delta),SIRT1 (sirtuin (silent mating type information regulation 2 homolog) 1(S. cerevisiae)), GNRH1 (gonadotropin-releasing hormone 1(luteinizing-releasing hormone)), PAPPA (pregnancy-associated plasmaprotein A, pappalysin 1), ARR3 (arrestin 3, retinal (X-arrestin)), NPPC(natriuretic peptide precursor C), AHSP (alpha hemoglobin stabilizingprotein), PTK2 (PTK2 protein tyrosine kinase 2), IL13 (interleukin 13),MTOR (mechanistic target of rapamycin (serine/threonine kinase)), ITGB2(integrin, beta 2 (complement component 3 receptor 3 and 4 subunit)),GSTT1 (glutathione S-transferase theta 1), IL6ST (interleukin 6 signaltransducer (gp130, oncostatin M receptor)), CPB2 (carboxypeptidase B2(plasma)), CYP1A2 (cytochrome P450, family 1, subfamily A, polypeptide2), HNF4A (hepatocyte nuclear factor 4, alpha), SLC6A4 (solute carrierfamily 6 (neurotransmitter transporter, serotonin), member 4), PLA2G6(phospholipase A2, group VI (cytosolic, calcium-independent)), TNFSF11(tumor necrosis factor (ligand) superfamily, member 11), SLC8A1 (solutecarrier family 8 (sodium/calcium exchanger), member 1), F2RL1(coagulation factor II (thrombin) receptor-like 1), AKR1A1 (aldo-ketoreductase family 1, member A1 (aldehyde reductase)), ALDH9A1 (aldehydedehydrogenase 9 family, member A1), BGLAP (bone gamma-carboxyglutamate(gla) protein), MTTP (microsomal triglyceride transfer protein), MTRR(5-methyltetrahydrofolate-homocysteine methyltransferase reductase),SULT1A3 (sulfotransferase family, cytosolic, 1A, phenol-preferring,member 3), RAGE (renal tumor antigen), C4B (complement component 4B(Chido blood group), P2RY12 (purinergic receptor P2Y, G-protein coupled,12), RNLS (renalase, FAD-dependent amine oxidase), CREB1 (cAMPresponsive element binding protein 1), POMC (proopiomelanocortin), RAC1(ras-related C3 botulinum toxin substrate 1 (rho family, small GTPbinding protein Racl)), LMNA (lamin NC), CD59 (CD59 molecule, complementregulatory protein), SCN5A (sodium channel, voltage-gated, type V, alphasubunit), CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide1), MIF (macrophage migration inhibitory factor(glycosylation-inhibiting factor)), MMP13 (matrix metallopeptidase 13(collagenase 3)), TIMP2 (TIMP metallopeptidase inhibitor 2), CYP19A1(cytochrome P450, family 19, subfamily A, polypeptide 1), CYP21A2(cytochrome P450, family 21, subfamily A, polypeptide 2), PTPN22(protein tyrosine phosphatase, non-receptor type 22 (lymphoid)), MYH14(myosin, heavy chain 14, non-muscle), MBL2 (mannose-binding lectin(protein C) 2, soluble (opsonic defect)), SELPLG (selectin P ligand),AOC3 (amine oxidase, copper containing 3 (vascular adhesion protein 1)),CTSL1 (cathepsin L1), PCNA (proliferating cell nuclear antigen), IGF2(insulin-like growth factor 2 (somatomedin A)), ITGB1 (integrin, beta 1(fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2,MSK12)), CAST (calpastatin), CXCL12 (chemokine (C—X—C motif) ligand 12(stromal cell-derived factor 1)), IGHE (immunoglobulin heavy constantepsilon), KCNE1 (potassium voltage-gated channel, Isk-related family,member 1), TFRC (transferrin receptor (p90, CD71)), COL1A1 (collagen,type I, alpha 1), COL1A2 (collagen, type I, alpha 2), IL2RB (interleukin2 receptor, beta), PLA2G10 (phospholipase A2, group X), ANGPT2(angiopoietin 2), PROCR (protein C receptor, endothelial (EPCR)), NOX4(NADPH oxidase 4), HAMP (hepcidin antimicrobial peptide), PTPN11(protein tyrosine phosphatase, non-receptor type 11), SLC2A1 (solutecarrier family 2 (facilitated glucose transporter), member 1), IL2RA(interleukin 2 receptor, alpha), CCL5 (chemokine (C—C motif) ligand 5),IRF1 (interferon regulatory factor 1), CFLAR (CASP8 and FADD-likeapoptosis regulator), CALCA (calcitonin-related polypeptide alpha),EIF4E (eukaryotic translation initiation factor 4E), GSTP1 (glutathioneS-transferase pi 1), JAK2 (Janus kinase 2), CYP3A5 (cytochrome P450,family 3, subfamily A, polypeptide 5), HSPG2 (heparan sulfateproteoglycan 2), CCL3 (chemokine (C—C motif) ligand 3), MYD88 (myeloiddifferentiation primary response gene (88)), VIP (vasoactive intestinalpeptide), SOAT1 (sterol O-acyltransferase 1), ADRBK1 (adrenergic, beta,receptor kinase 1), NR4A2 (nuclear receptor subfamily 4, group A, member2), MMP8 (matrix metallopeptidase 8 (neutrophil collagenase)), NPR2(natriuretic peptide receptor B/guanylate cyclase B (atrionatriureticpeptide receptor B)), GCH1 (GTP cyclohydrolase 1), EPRS(glutamyl-prolyl-tRNA synthetase), PPARGC1A (peroxisomeproliferator-activated receptor gamma, coactivator 1 alpha), F12(coagulation factor XII (Hageman factor)), PECAM1 (platelet/endothelialcell adhesion molecule), CCL4 (chemokine (C—C motif) ligand 4), SERPINA3(serpin peptidase inhibitor, clade A (alpha-1 antiproteinase,antitrypsin), member 3), CASR (calcium-sensing receptor), GJA5 (gapjunction protein, alpha 5, 40 kDa), FABP2 (fatty acid binding protein 2,intestinal), TTF2 (transcription termination factor, RNA polymerase II),PROS1 (protein S (alpha)), CTF1 (cardiotrophin 1), SGCB (sarcoglycan,beta (43 kDa dystrophin-associated glycoprotein)), YME1L1 (YME1-like 1(S. cerevisiae)), CAMP (cathelicidin antimicrobial peptide), ZC3H12A(zinc finger CCCH-type containing 12A), AKR1B1 (aldo-keto reductasefamily 1, member B1 (aldose reductase)), DES (desmin), MMP7 (matrixmetallopeptidase 7 (matrilysin, uterine)), AHR (aryl hydrocarbonreceptor), CSF1 (colony stimulating factor 1 (macrophage)), HDAC9(histone deacetylase 9), CTGF (connective tissue growth factor), KCNMA1(potassium large conductance calcium-activated channel, subfamily M,alpha member 1), UGT1A (UDP glucuronosyltransferase 1 family,polypeptide A complex locus), PRKCA (protein kinase C, alpha), COMT(catechol-.beta.-methyltransferase), S100B (S100 calcium binding proteinB), EGR1 (early growth response 1), PRL (prolactin), IL15 (interleukin15), DRD4 (dopamine receptor D4), CAMK2G (calcium/calmodulin-dependentprotein kinase II gamma), SLC22A2 (solute carrier family 22 (organiccation transporter), member 2), CCL11 (chemokine (C—C motif) ligand 11),PGF (B321 placental growth factor), THPO (thrombopoietin), GP6(glycoprotein VI (platelet)), TACR1 (tachykinin receptor 1), NTS(neurotensin), HNF1A (HNF1 homeobox A), SST (somatostatin), KCND1(potassium voltage-gated channel, Shal-related subfamily, member 1),LOC646627 (phospholipase inhibitor), TBXAS1 (thromboxane A synthase 1(platelet)), CYP2J2 (cytochrome P450, family 2, subfamily J, polypeptide2), TBXA2R (thromboxane A2 receptor), ADH1C (alcohol dehydrogenase 1C(class I), gamma polypeptide), ALOX12 (arachidonate 12-lipoxygenase),AHSG (alpha-2-HS-glycoprotein), BHMT (betaine-homocysteinemethyltransferase), GJA4 (gap junction protein, alpha 4, 37 kDa),SLC25A4 (solute carrier family 25 (mitochondrial carrier; adeninenucleotide translocator), member 4), ACLY (ATP citrate lyase), ALOX5AP(arachidonate 5-lipoxygenase-activating protein), NUMA1 (nuclear mitoticapparatus protein 1), CYP27B1 (cytochrome P450, family 27, subfamily B,polypeptide 1), CYSLTR2 (cysteinyl leukotriene receptor 2), SOD3(superoxide dismutase 3, extracellular), LTC4S (leukotriene C4synthase), UCN (urocortin), GHRL (ghrelin/obestatin prepropeptide),APOC2 (apolipoprotein C-II), CLEC4A (C-type lectin domain family 4,member A), KBTBD10 (kelch repeat and BTB (POZ) domain containing 10),TNC (tenascin C), TYMS (thymidylate synthetase), SHCI (SHC (Src homology2 domain containing) transforming protein 1), LRP1 (low densitylipoprotein receptor-related protein 1), SOCS3 (suppressor of cytokinesignaling 3), ADH1B (alcohol dehydrogenase 1B (class I), betapolypeptide), KLK3 (kallikrein-related peptidase 3), HSD11B1(hydroxysteroid (11-beta) dehydrogenase 1), VKORC1 (vitamin K epoxidereductase complex, subunit 1), SERPINB2 (serpin peptidase inhibitor,clade B (ovalbumin), member 2), TNS1 (tensin 1), RNF19A (ring fingerprotein 19A), EPOR (erythropoietin receptor), ITGAM (integrin, alpha M(complement component 3 receptor 3 subunit)), PITX2 (paired-likehomeodomain 2), MAPK7 (mitogen-activated protein kinase 7), FCGR3A (Fcfragment of IgG, low affinity 111a, receptor (CD16a)), LEPR (leptinreceptor), ENG (endoglin), GPX1 (glutathione peroxidase 1), GOT2(glutamic-oxaloacetic transaminase 2, mitochondrial (aspartateaminotransferase 2)), HRH1 (histamine receptor H1), NR112 (nuclearreceptor subfamily 1, group I, member 2), CRH (corticotropin releasinghormone), HTR1A (5-hydroxytryptamine (serotonin) receptor 1A), VDAC1(voltage-dependent anion channel 1), HPSE (heparanase), SFTPD(surfactant protein D), TAP2 (transporter 2, ATP-binding cassette,sub-family B (MDR/TAP)), RNF123 (ring finger protein 123), PTK2B (PTK2Bprotein tyrosine kinase 2 beta), NTRK2 (neurotrophic tyrosine kinase,receptor, type 2), IL6R (interleukin 6 receptor), ACHE(acetylcholinesterase (Yt blood group)), GLP1R (glucagon-like peptide 1receptor), GHR (growth hormone receptor), GSR (glutathione reductase),NQO1 (NAD(P)H dehydrogenase, quinone 1), NR5A1 (nuclear receptorsubfamily 5, group A, member 1), GJB2 (gap junction protein, beta 2, 26kDa), SLC9A1 (solute carrier family 9 (sodium/hydrogen exchanger),member 1), MAOA (monoamine oxidase A), PCSK9 (proprotein convertasesubtilisin/kexin type 9), FCGR2A (Fc fragment of IgG, low affinity Ha,receptor (CD32)), SERPINF1 (serpin peptidase inhibitor, clade F (alpha-2antiplasmin, pigment epithelium derived factor), member 1), EDN3(endothelin 3), DHFR (dihydrofolate reductase), GAS6 (growtharrest-specific 6), SMPD1 (sphingomyelin phosphodiesterase 1, acidlysosomal), UCP2 (uncoupling protein 2 (mitochondrial, proton carrier)),TFAP2A (transcription factor AP-2 alpha (activating enhancer bindingprotein 2 alpha)), C4BPA (complement component 4 binding protein,alpha), SERPINF2 (serpin peptidase inhibitor, clade F (alpha-2antiplasmin, pigment epithelium derived factor), member 2), TYMP(thymidine phosphorylase), ALPP (alkaline phosphatase, placental (Reganisozyme)), CXCR2 (chemokine (C—X—C motif) receptor 2), SLC39A3 (solutecarrier family 39 (zinc transporter), member 3), ABCG2 (ATP-bindingcassette, sub-family G (WHITE), member 2), ADA (adenosine deaminase),JAK3 (Janus kinase 3), HSPA1A (heat shock 70 kDa protein 1A), FASN(fatty acid synthase), FGF1 (fibroblast growth factor 1 (acidic)), F11(coagulation factor XI), ATP7A (ATPase, Cu++transporting, alphapolypeptide), CR1 (complement component (3b/4b) receptor 1 (Knops bloodgroup)), GFAP (glial fibrillary acidic protein), ROCK1 (Rho-associated,coiled-coil containing protein kinase 1), MECP2 (methyl CpG bindingprotein 2 (Rett syndrome)), MYLK (myosin light chain kinase), BCHE(butyrylcholinesterase), LIPE (lipase, hormone-sensitive), PRDX5(peroxiredoxin 5), ADORA1 (adenosine A1 receptor), WRN (Werner syndrome,RecQ helicase-like), CXCR3 (chemokine (C—X—C motif) receptor 3), CD81(CD81 molecule), SMAD7 (SMAD family member 7), LAMC2 (laminin, gamma 2),MAP3K5 (mitogen-activated protein kinase 5), CHGA (chromogranin A(parathyroid secretory protein 1)), IAPP (islet amyloid polypeptide),RHO (rhodopsin), ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase1), PTHLH (parathyroid hormone-like hormone), NRG1 (neuregulin 1), VEGFC(vascular endothelial growth factor C), ENPEP (glutamyl aminopeptidase(aminopeptidase A)), CEBPB (CCAAT/enhancer binding protein (C/EBP),beta), NAGLU (N-acetylglucosaminidase, alpha-), F2RL3 (coagulationfactor II (thrombin) receptor-like 3), CX3CL1 (chemokine (C-X3-C motif)ligand 1), BDKRB1 (bradykinin receptor B1), ADAMTS13 (ADAMmetallopeptidase with thrombospondin type 1 motif, 13), ELANE (elastase,neutrophil expressed), ENPP2 (ectonucleotidepyrophosphatase/phosphodiesterase 2), CISH (cytokine inducibleSH2-containing protein), GAST (gastrin), MYOC (myocilin, trabecularmeshwork inducible glucocorticoid response), ATP1A2 (ATPase,Na+/K+transporting, alpha 2 polypeptide), NF1 (neurofibromin 1), GJB1(gap junction protein, beta 1, 32 kDa), MEF2A (myocyte enhancer factor2A), VCL (vinculin), BMPR2 (bone morphogenetic protein receptor, type II(serine/threonine kinase)), TUBB (tubulin, beta), CDC42 (cell divisioncycle 42 (GTP binding protein, 25 kDa)), KRT18 (keratin 18), HSF1 (heatshock transcription factor 1), MYB (v-myb myeloblastosis viral oncogenehomolog (avian)), PRKAA2 (protein kinase, AMP-activated, alpha 2catalytic subunit), ROCK2 (Rho-associated, coiled-coil containingprotein kinase 2), TFPI (tissue factor pathway inhibitor(lipoprotein-associated coagulation inhibitor)), PRKG1 (protein kinase,cGMP-dependent, type I), BMP2 (bone morphogenetic protein 2), CTNND1(catenin (cadherin-associated protein), delta 1), CTH (cystathionase(cystathionine gamma-lyase)), CTSS (cathepsin S), VAV2 (vav 2 guaninenucleotide exchange factor), NPY2R (neuropeptide Y receptor Y2), IGFBP2(insulin-like growth factor binding protein 2, 36 kDa), CD28 (CD28molecule), GSTA1 (glutathione S-transferase alpha 1), PPIA(peptidylprolyl isomerase A (cyclophilin A)), APOH (apolipoprotein H(beta-2-glycoprotein I)), S100A8 (S100 calcium binding protein A8), I11(interleukin 11), ALOX15 (arachidonate 15-lipoxygenase), FBLN1 (fibulin1), NR1H3 (nuclear receptor subfamily 1, group H, member 3), SCD(stearoyl-CoA desaturase (delta-9-desaturase)), GIP (gastric inhibitorypolypeptide), CHGB (chromogranin B (secretogranin 1)), PRKCB (proteinkinase C, beta), SRD5A1 (steroid-5-alpha-reductase, alpha polypeptide 1(3-oxo-5 alpha-steroid delta 4-dehydrogenase alpha 1)), HSD11B2(hydroxysteroid (11-beta) dehydrogenase 2), CALCRL (calcitoninreceptor-like), GALNT2 (UDP-N-acetyl-alpha-D-galactosamine:polypeptideN-acetylgalactosaminyltransferase 2 (GalNAc-T2)), ANGPTL4(angiopoietin-like 4), KCNN4 (potassium intermediate/small conductancecalcium-activated channel, subfamily N, member 4), PIK3C2A(phosphoinositide-3-kinase, class 2, alpha polypeptide), HBEGF(heparin-binding EGF-like growth factor), CYP7A1 (cytochrome P450,family 7, subfamily A, polypeptide 1), HLA-DRB5 (majorhistocompatibility complex, class II, DR beta 5), BNIP3 (BCL2/adenovirusE1B 19 kDa interacting protein 3), GCKR (glucokinase (hexokinase 4)regulator), S100A12 (S100 calcium binding protein A12), PADI4 (peptidylarginine deiminase, type IV), HSPA14 (heat shock 70 kDa protein 14),CXCR1 (chemokine (C—X—C motif) receptor 1), H19 (H19, imprintedmaternally expressed transcript (non-protein coding)), KRTAP19-3(keratin associated protein 19-3), IDDM2 (insulin-dependent diabetesmellitus 2), RAC2 (ras-related C3 botulinum toxin substrate 2 (rhofamily, small GTP binding protein Rac2)), RYR1 (ryanodine receptor 1(skeletal)), CLOCK (clock homolog (mouse)), NGFR (nerve growth factorreceptor (TNFR superfamily, member 16)), DBH (dopamine beta-hydroxylase(dopamine beta-monooxygenase)), CHRNA4 (cholinergic receptor, nicotinic,alpha 4), CACNA1C (calcium channel, voltage-dependent, L type, alpha 1Csubunit), PRKAG2 (protein kinase, AMP-activated, gamma 2 non-catalyticsubunit), CHAT (choline acetyltransferase), PTGDS (prostaglandin D2synthase 21 kDa (brain)), NR1H2 (nuclear receptor subfamily 1, group H,member 2), TEK (TEK tyrosine kinase, endothelial), VEGFB (vascularendothelial growth factor B), MEF2C (myocyte enhancer factor 2C),MAPKAPK2 (mitogen-activated protein kinase-activated protein kinase 2),TNFRSF11A (tumor necrosis factor receptor superfamily, member 11a, NFKBactivator), HSPA9 (heat shock 70 kDa protein 9 (mortalin)), CYSLTR1(cysteinyl leukotriene receptor 1), MAT1A (methionineadenosyltransferase I, alpha), OPRL1 (opiate receptor-like 1), IMPA1(inositol(myo)-1(or 4)-monophosphatase 1), CLCN2 (chloride channel 2),DLD (dihydrolipoamide dehydrogenase), PSMA6 (proteasome (prosome,macropain) subunit, alpha type, 6), PSMB8 (proteasome (prosome,macropain) subunit, beta type, 8 (large multifunctional peptidase 7)),CHI3L1 (chitinase 3-like 1 (cartilage glycoprotein-39)), ALDH1B1(aldehyde dehydrogenase 1 family, member B1), PARP2 (poly (ADP-ribose)polymerase 2), STAR (steroidogenic acute regulatory protein), LBP(lipopolysaccharide binding protein), ABCC6 (ATP-binding cassette,sub-family C(CFTR/MRP), member 6), RGS2 (regulator of G-proteinsignaling 2, 24 kDa), EFNB2 (ephrin-B2), GJB6 (gap junction protein,beta 6, 30 kDa), APOA2 (apolipoprotein A-II), AMPD1 (adenosinemonophosphate deaminase 1), DYSF (dysferlin, limb girdle musculardystrophy 2B (autosomal recessive)), FDFT1 (farnesyl-diphosphatefarnesyltransferase 1), EDN2 (endothelin 2), CCR6 (chemokine (C—C motif)receptor 6), GJB3 (gap junction protein, beta 3, 31 kDa), ILIRL1(interleukin 1 receptor-like 1), ENTPD1 (ectonucleoside triphosphatediphosphohydrolase 1), BBS4 (Bardet-Biedl syndrome 4), CELSR2 (cadherin,EGF LAG seven-pass G-type receptor 2 (flamingo homolog, Drosophila)),F1IR (F11 receptor), RAPGEF3 (Rap guanine nucleotide exchange factor(GEF) 3), HYAL1 (hyaluronoglucosaminidase 1), ZNF259 (zinc fingerprotein 259), ATOX1 (ATX1 antioxidant protein 1 homolog (yeast)), ATF6(activating transcription factor 6), KHK (ketohexokinase(fructokinase)), SAT1 (spermidine/spermine N1-acetyltransferase 1), GGH(gamma-glutamyl hydrolase (conjugase, folylpolygammaglutamylhydrolase)), TIMP4 (TIMP metallopeptidase inhibitor 4), SLC4A4 (solutecarrier family 4, sodium bicarbonate cotransporter, member 4), PDE2A(phosphodiesterase 2A, cGMP-stimulated), PDE3B (phosphodiesterase 3B,cGMP-inhibited), FADS1 (fatty acid desaturase 1), FADS2 (fatty aciddesaturase 2), TMSB4X (thymosin beta 4, X-linked), TXNIP (thioredoxininteracting protein), LIMS1 (LIM and senescent cell antigen-like domains1), RHOB (ras homolog gene family, member B), LY96 (lymphocyte antigen96), FOXO1 (forkhead box 01), PNPLA2 (patatin-like phospholipase domaincontaining 2), TRH (thyrotropin-releasing hormone), GJC1 (gap junctionprotein, gamma 1, 45 kDa), SLC17A5 (solute carrier family 17(anion/sugar transporter), member 5), FTO (fat mass and obesityassociated), GJD2 (gap junction protein, delta 2, 36 kDa), PSRC1(proline/serine-rich coiled-coil 1), CASP12 (caspase 12(gene/pseudogene)), GPBAR1 (G protein-coupled bile acid receptor 1), PXK(PX domain containing serine/threonine kinase), IL33 (interleukin 33),TRIB1 (tribbles homolog 1 (Drosophila)), PBX4 (pre-B-cell leukemiahomeobox 4), NUPR1 (nuclear protein, transcriptional regulator, 1),15-Sep(15 kDa selenoprotein), CILP2 (cartilage intermediate layerprotein 2), TERC (telomerase RNA component), GGT2(gamma-glutamyltransferase 2), MT-CO1 (mitochondrially encodedcytochrome c oxidase I), and UOX (urate oxidase, pseudogene). Any ofthese sequences, may be a target for the CRISPR-Cas system, e.g., toaddress mutation.

In an additional embodiment, the chromosomal sequence may further beselected from Pon1 (paraoxonase 1), LDLR (LDL receptor), ApoE(Apolipoprotein E), Apo B-100 (Apolipoprotein B-100), ApoA(Apolipoprotein(a)), ApoA1 (Apolipoprotein A1), CBS (CystathionineB-synthase), Glycoprotein IIb/IIb, MTHRF (5,10-methylenetetrahydrofolatereductase (NADPH), and combinations thereof. In one iteration, thechromosomal sequences and proteins encoded by chromosomal sequencesinvolved in cardiovascular disease may be chosen from Cacna1C, Sod1,Pten, Ppar(alpha), Apo E, Leptin, and combinations thereof as target(s)for the CRISPR-Cas system.

Treating Diseases of the Liver and Kidney

The present invention also contemplates delivering the CRISPR-Cas systemdescribed herein, e.g. C2c1 or C2c3 effector protein systems, to theliver and/or kidney. Delivery strategies to induce cellular uptake ofthe therapeutic nucleic acid include physical force or vector systemssuch as viral-, lipid- or complex-based delivery, or nanocarriers. Fromthe initial applications with less possible clinical relevance, whennucleic acids were addressed to renal cells with hydrodynamic highpressure injection systemically, a wide range of gene therapeutic viraland non-viral carriers have been applied already to targetposttranscriptional events in different animal kidney disease models invivo (Csaba Révész and Péter Hamar (2011). Delivery Methods to TargetRNAs in the Kidney, Gene Therapy Applications, Prof. Chunsheng Kang(Ed.), ISBN: 978-953-307-541-9, InTech, Available from:www.intechopen.com/books/gene-therapy-appiications/delivery-methods-to-target-rnas-inthe-kidney).Delivery methods to the kidney may include those in Yuan et al. (Am JPhysiol Renal Physiol 295: F605-F617, 2008) investigated whether in vivodelivery of small interfering RNAs (siRNAs) targeting the12/15-lipoxygenase (12/15-LO) pathway of arachidonate acid metabolismcan ameliorate renal injury and diabetic nephropathy (DN) in astreptozotocininjected mouse model of type 1 diabetes. To achievegreater in vivo access and siRNA expression in the kidney, Yuan et al.used double-stranded 12/15-LO siRNA oligonucleotides conjugated withcholesterol. About 400 μg of siRNA was injected subcutaneously intomice. The method of Yuang et al. may be applied to the CRISPR Cas systemof the present invention contemplating a 1-2 g subcutaneous injection ofCRISPR Cas conjugated with cholesterol to a human for delivery to thekidneys.

Molitoris et al. (J Am Soc Nephrol 20: 1754-1764, 2009) exploitedproximal tubule cells (PTCs), as the site of oligonucleotidereabsorption within the kidney to test the efficacy of siRNA targeted top53, a pivotal protein in the apoptotic pathway, to prevent kidneyinjury. Naked synthetic siRNA to p53 injected intravenously 4 h afterischemic injury maximally protected both PTCs and kidney function.Molitoris et al.'s data indicates that rapid delivery of siRNA toproximal tubule cells follows intravenous administration. Fordose-response analysis, rats were injected with doses of siP53, 0.33; 1,3, or 5 mg/kg, given at the same four time points, resulting incumulative doses of 1.32; 4, 12, and 20 mg/kg, respectively. All siRNAdoses tested produced a SCr reducing effect on day one with higher dosesbeing effective over approximately five days compared with PBS-treatedischemic control rats. The 12 and 20 mg/kg cumulative doses provided thebest protective effect. The method of Molitoris et al. may be applied tothe nucleic acid-targeting system of the present invention contemplating12 and 20 mg/kg cumulative doses to a human for delivery to the kidneys.

Thompson et al. (Nucleic Acid Therapeutics, Volume 22, Number 4, 2012)reports the toxicological and pharmacokinetic properties of thesynthetic, small interfering RNA I5NP following intravenousadministration in rodents and nonhuman primates. I5NP is designed to actvia the RNA interference (RNAi) pathway to temporarily inhibitexpression of the pro-apoptotic protein p53 and is being developed toprotect cells from acute ischemia/reperfusion injuries such as acutekidney injury that can occur during major cardiac surgery and delayedgraft function that can occur following renal transplantation. Doses of800 mg/kg I5NP in rodents, and 1,000 mg/kg I5NP in nonhuman primates,were required to elicit adverse effects, which in the monkey wereisolated to direct effects on the blood that included a sub-clinicalactivation of complement and slightly increased clotting times. In therat, no additional adverse effects were observed with a rat analogue ofI5NP, indicating that the effects likely represent class effects ofsynthetic RNA duplexes rather than toxicity related to the intendedpharmacologic activity of I5NP. Taken together, these data supportclinical testing of intravenous administration of I5NP for thepreservation of renal function following acute ischemia/reperfusioninjury. The no observed adverse effect level (NOAEL) in the monkey was500 mg/kg. No effects on cardiovascular, respiratory, and neurologicparameters were observed in monkeys following i.v. administration atdose levels up to 25 mg/kg. Therefore, a similar dosage may becontemplated for intravenous administration of CRISPR Cas to the kidneysof a human.

Shimizu et al. (J Am Soc Nephrol 21: 622-633, 2010) developed a systemto target delivery of siRNAs to glomeruli via poly(ethyleneglycol)-poly(L-lysine)-based vehicles. The siRNA/nanocarrier complex wasapproximately 10 to 20 nm in diameter, a size that would allow it tomove across the fenestrated endothelium to access to the mesangium.After intraperitoneal injection of fluorescence-labeledsiRNA/nanocarrier complexes, Shimizu et al. detected siRNAs in the bloodcirculation for a prolonged time. Repeated intraperitonealadministration of a mitogen-activated protein kinase 1 (MAPK1)siRNA/nanocarrier complex suppressed glomerular MAPK1 mRNA and proteinexpression in a mouse model of glomerulonephritis. For the investigationof siRNA accumulation, Cy5-labeled siRNAs complexed with PICnanocarriers (0.5 ml, 5 nmol of siRNA content), naked Cy5-labeled siRNAs(0.5 ml, 5 nmol), or Cy5-labeled siRNAs encapsulated in HVJ-E (0.5 ml, 5nmol of siRNA content) were administrated to BALBc mice. The method ofShimizu et al. may be applied to the nucleic acid-targeting system ofthe present invention contemplating a dose of about of 10-20 μmol CRISPRCas complexed with nanocarriers in about 1-2 liters to a human forintraperitoneal administration and delivery to the kidneys.

TABLE 9 Delivery methods to the kidney are summarazied as follows:Delivery Functional method Carrier Target RNA Disease Model assaysAuthor Hydrodynamic/ TransIT In Vivo p85α Acute renal Ischemia- Uptake,Larson et al., Lipid Gene Delivery injury reperfusion biodistributionSurgery, (August System, DOTAP 2007), Vol. 142, No. 2, pp. (262-269)Hydrodynamic/ Lipofectamine Fas Acute renal Ischemia- Blood urea Hamaret al., Lipid 2000 injury reperfusion nitrogen, Fas Proc Natl AcadImmunohistochemistry, Sci, (October 2004), apoptosis, Vol. 101, No. 41,histological pp. (14883-14888) scoring Hydrodynamic n.a. Apoptosis Acuterenal Ischemia- n.a. Zheng et al., Am cascade injury reperfusion JPathol, (October elements 2008), Vol. 173, No. 4, pp. (973-980)Hydrodynamic n.a. Nuclear Acute renal Ischemia- n.a. Feng et al., factorkappa- injury reperfusion Transplantation, b (NFkB) (May 2009), Vol. 87,No. 9, pp. (1283-1289) Hydrodynamic/ Lipofectamine Apoptosis Acute renalIschemia- Apoptosis, Xie & Guo, Am Viral 2000 antagonizing injuryreperfusion oxidative stress, Soc Nephrol, transcription caspase(December 2006), Vol. factor activation, 17, No. 12, pp. (AATF) membranelipid (3336-3346) peroxidation Hydrodynamic pBAsi mU6 Neo/ GremlinDiabetic Streptozotozin- Proteinuria, Q. Zhang et al., TransIT-EEnephropathy induced serum creatinine, PloS ONE, (July Hydrodynamicdiabetes glomerular and 2010), Vol. 5, Delivery System tubular diameter,No. 7, e11709, collagen type pp. (1-13) IV/BMP7 expression Viral/LipidpSUPER TGF-β type Interstitial Unilateral α-SMA Kushibikia et al.,vector/ II receptor renal urethral expression, J ControlledLipofectamine fibrosis obstruction collagen content, Release, (July2005), Vol. 105, No. 3, pp. (318-331) Viral Adeno-associated MineralHypertension Cold- blood pressure, Wang et al., Gene virus-2 corticoidcaused renal induced serum albumin, Therapy, (July receptor damagehypertension serum urea 2006), Vol. 13, nitrogen, serum No. 14, pp.creatinine, (1097-1103) kidney weight, urinary sodium Hydrodynamic/ pU6vector Luciferase n.a. n.a. uptake Kobayashi et al., Viral Journal ofPharmacology and Experimental Therapeutics, (February 2004), Vol. 308,No. 2, pp. (688-693) Lipid Lipoproteins, apoB1, apoM n.a. n.a. Uptake,binding Wolfrum et al., albumin affinity to Nature lipoproteins andBiotechnology, albumin (September 2007), Vol. 25, No. 10, pp.(1149-1157) Lipid Lipofectamine2000 p53 Acute renal IschemicHistological Molitoris et al., J injury and scoring, Am Soc Nephrol,cisplatin- apoptosis (August 2009), Vol. induced 20, No. 8, acute injurypp. (1754-1764) Lipid DOTAP/DOPE, COX-2 Breast MDA-MB- Cell viability,Mikhaylova et al., DOTAP/DOPE/ adeno- 231 breast uptake Cancer GeneDOPE- carcinoma cancer Therapy, (March PEG2000 xenograft- 2011), Vol.16, No. bearing 3, pp. (217-226) mouse Lipid Cholesterol 12/15- DiabeticStreptozotocin- Albuminuria, Yuan et al., Am J lipoxygenase nephropathyinduced urinary Physiol Renal diabetes creatinine, Physiol, (Junehistology, type I 2008), Vol. 295, and IV collagen, pp. (F605-F617)TGF-β, fibronectin, plasminogen activator inhibitor 1 LipidLipofectamine Mitochondrial Diabetic Streptozotocin- Cell proliferationY. Zhang et al., J 2000 membrane nephropathy induced and apoptosis, AmSoc Nephrol, 44 (TIM44) diabetes histology, ROS, (April 2006), Vol.mitochondrial 17, No. 4, pp. import of Mn- (1090-1101) SOD andglutathione peroxidase, cellular membrane polarization Hydrodynamic/Proteoliposome RLIP76 Renal Caki-2 uptake Singhal et al., Lipidcarcinoma kidney Cancer Res, cancer (May 2009), Vol. xenograft- 69, No.10, pp. bearing (4244-4251) mouse Polymer PEGylated PEI Luciferase n.a.n.a. Uptake, Malek et al., pGL3 biodistribution, Toxicology anderythrocyte Applied aggregation Pharmacology, (April 2009), Vol. 236,No. 1, pp. (97-108) Polymer PEGylated MAPK1 Lupus Glomerulo-Proteinuria, Shimizu et al., J poly-L-lysine glomerulo- nephritisglomerulosclerosis, Am Soc nephritis TGF-β, Nephrology, (Aprilfibronectin, 2010), Vol. 21, plasminogen No. 4, pp. (622-633) activatorinhibitor 1 Polymer/Nano Hyaluronic acid/ VEGF Kidney B16F1Biodistribution, Jiang et al., particle Quantum dot/ cancer/ melanomacitotoxicity, Molecular PEI melanoma tumor- tumor volume, Pharmaceutics,bearing endocytosis (May-June 2009), mouse Vol. 6, No. 3, pp. (727-737)Polymer/Nano PEGylated GAPDH n.a. n.a. cell viability, Cao et al, Jparticle polycaprolactone uptake Controlled nanofiber Release, (June2010), Vol. 144, No. 2, pp. (203-212) Aptamer Spiegelmer CCGlomerulosclerosis Uninephrectomized urinary albumin, Ninichuk et al.,mNOX-E36 chemokine mouse urinary Am J Pathol, ligand 2 creatinine,(March 2008), Vol. histopathology, 172, No. 3, pp. glomerular (628-637)filtration rate, macrophage count, serum Ccl2, Mac-2+, Ki-67+ AptamerAptamer NOX- vasopressin Congestive n.a. Binding affinity Purschke etal., F37 (AVP) heart failure to D-AVP, Proc Natl Acad Inhibition of Sci,(March 2006), AVP Signaling, Vol. 103, No. 13, Urine osmolality pp.(5173-5178) and sodium concentration,Targeting the Liver or Liver Cells

Targeting liver cells is provided. This may be in vitro or in vivo.Hepatocytes are preferred. Delivery of the CRISPR protein, such as C2c1or C2c3 herein may be via viral vectors, especially AAV (and inparticular AAV2/6) vectors. These may be administered by intravenousinjection.

A preferred target for liver, whether in vitro or in vivo, is thealbumin gene. This is a so-called ‘safe harbor” as albumin is expressedat very high levels and so some reduction in the production of albuminfollowing successful gene editing is tolerated. It is also preferred asthe high levels of expression seen from the albumin promoter/enhancerallows for useful levels of correct or transgene production (from theinserted donor template) to be achieved even if only a small fraction ofhepatocytes are edited.

Intron 1 of albumin has been shown by Wechsler et al. (reported at the57th Annual Meeting and Exposition of the American Society ofHematology—abstract available online atash.confex.com/ash/2015/webprogram/Paper86495.html and presented on 6thDecember 2015) to be a suitable target site. Their work used Zn Fingersto cut the DNA at this target site, and suitable guide sequences can begenerated to guide cleavage at the same site by a CRISPR protein.

The use of targets within highly-expressed genes (genes with highlyactive enhancers/promoters) such as albumin may also allow apromoterless donor template to be used, as reported by Wechsler et al.and this is also broadly applicable outside liver targeting. Otherexamples of highly-expressed genes are known.

Liver-Associated Blood Disorders. Especially Hemophilia and inParticular Hemophilia B

Successful gene editing of hepatocytes has been achieved in mice (bothin vitro and in vivo) and in non-human primates (in vivo), showing thattreatment of blood disorders through gene editing/genome engineering inhepatocytes is feasible. In particular, expression of the human F9 (hF9)gene in hepatocytes has been shown in non-human primates indicating atreatment for Hemophilia B in humans.

Wechsler et al. reported at the 57th Annual Meeting and Exposition ofthe American Society of Hematology (abstract presented 6th December 2015and available online atash.confex.com/ash/2015/webprogram/Paper86495.html) that they hassuccessfully expressed human F9 (hF9) from hepatocytes in non-humanprimates through in vivo gene editing. This was achieved using 1) twozinc finger nucleases (ZFNs) targeting intron 1 of the albumin locus,and 2) a human F9 donor template construct. The ZFNs and donor templatewere encoded on separate hepatotropic adeno-associated virus serotype2/6 (AAV2/6) vectors injected intravenously, resulting in targetedinsertion of a corrected copy of the hF9 gene into the albumin locus ina proportion of liver hepatocytes.

The albumin locus was selected as a “safe harbor” as production of thismost abundant plasma protein exceeds 10 g/day, and moderate reductionsin those levels are well-tolerated. Genome edited hepatocytes producednormal hFIX (hF9) in therapeutic quantities, rather than albumin, drivenby the highly active albumin enhancer/promoter. Targeted integration ofthe hF9 transgene at the albumin locus and splicing of this gene intothe albumin transcript was shown.

Mice studies: C57BL/6 mice were administered vehicle (n=20) or AAV2/6vectors (n=25) encoding mouse surrogate reagents at 1.0×1013 vectorgenome (vg)/kg via tail vein injection. ELISA analysis of plasma hFIX inthe treated mice showed peak levels of 50-1053 ng/mL that were sustainedfor the duration of the 6-month study. Analysis of FIX activity frommouse plasma confirmed bioactivity commensurate with expression levels.

Non-human primate (NHP) studies: a single intravenous co-infusion ofAAV2/6 vectors encoding the NHP targeted albumin-specific ZFNs and ahuman F9 donor at 1.2×1013 vg/kg (n=5/group) resulted in >50 ng/mL (>1%of normal) in this large animal model. The use of higher AAV2/6 doses(up to 1.5×1014 vg/kg) yielded plasma hFIX levels up to 1000 ng/ml (or20% of normal) in several animals and up to 2000 ng/ml (or 50% ofnormal) in a single animal, for the duration of the study (3 months).

The treatment was well tolerated in mice and NHPs, with no significanttoxicological findings related to AAV2/6 ZFN+donor treatment in eitherspecies at therapeutic doses. Sangamo (CA, USA) has since applied to theFDA, and been granted, permission to conduct the world's first humanclinical trial for an in vivo genome editing application. This followson the back of the EMEA's approval of the Glybera gene therapy treatmentof lipoprotein lipase deficiency.

Accordingly, it is preferred, in some embodiments, that any or all ofthe following are used:

-   -   AAV (especially AAV2/6) vectors, preferably administered by        intravenous injection;    -   Albumin as target for gene editing/insertion of        transgene/template-especially at intron 1 of albumin;    -   human F9 donor template; and/or    -   a promoterless donor template.        Hemophilia B

Accordingly, in some embodiments, it is preferred that the presentinvention is used to treat Hemophilia B. As such it is preferred that atemplate is provided and that this is the human F9 gene. It will beappreciated that the hF9 template comprises the wt or ‘correct’ versionof hF9 so that the treatment is effective.

In an alternative embodiment, the hemophilia B version of F9 may bedelivered so as to create a model organism, cell or cell line (forexample a murine or non-human primate model organism, cell or cellline), the model organism, cell or cell line having or carrying theHemophilia B phenotype, i.e. an inability to produce wt F9.

Hemophilia A

In some embodiments, the F9 (factor IX) gene may be replaced by the F8(factor VIII) gene described above, leading to treatment of Hemophilia A(through provision of a correct F8 gene) and/or creation of a HemophiliaA model organism, cell or cell line (through provision of an incorrect,Hemophilia A version of the F8 gene).

Hemophilia C

In some embodiments, the F9 (factor IX) gene may be replaced by the F11(factor XI) gene described above, leading to treatment of Hemophilia C(through provision of a correct F11 gene) and/or creation of aHemophilia C model organism, cell or cell line (through provision of anincorrect, Hemophilia C version of the F11 gene).

Treating Epithelial and Lung Diseases

The present invention also contemplates delivering the CRISPR-Cas systemdescribed herein, e.g. C2c1 or C2c3 effector protein systems, to one orboth lungs.

Although AAV-2-based vectors were originally proposed for CFTR deliveryto CF airways, other serotypes such as AAV-1, AAV-5, AAV-6, and AAV-9exhibit improved gene transfer efficiency in a variety of models of thelung epithelium (see, e.g., Li et al., Molecular Therapy, vol. 17 no.12, 2067-277 Dec. 2009). AAV-1 was demonstrated to be ˜100-fold moreefficient than AAV-2 and AAV-5 at transducing human airway epithelialcells in vitro, 5 although AAV-1 transduced murine tracheal airwayepithelia in vivo with an efficiency equal to that of AAV-5. Otherstudies have shown that AAV-5 is 50-fold more efficient than AAV-2 atgene delivery to human airway epithelium (HAE) in vitro andsignificantly more efficient in the mouse lung airway epithelium invivo. AAV-6 has also been shown to be more efficient than AAV-2 in humanairway epithelial cells in vitro and murine airways in vivo. 8 The morerecent isolate, AAV-9, was shown to display greater gene transferefficiency than AAV-5 in murine nasal and alveolar epithelia in vivowith gene expression detected for over 9 months suggesting AAV mayenable long-term gene expression in vivo, a desirable property for aCFTR gene delivery vector. Furthermore, it was demonstrated that AAV-9could be readministered to the murine lung with no loss of CFTRexpression and minimal immune consequences. CF and non-CF HAE culturesmay be inoculated on the apical surface with 100 μl of AAV vectors forhours (see, e.g., Li et al., Molecular Therapy, vol. 17 no. 12, 2067-277Dec. 2009). The MOI may vary from 1×10³ to 4×10⁵ vector genomes/cell,depending on virus concentration and purposes of the experiments. Theabove cited vectors are contemplated for the delivery and/oradministration of the invention.

Zamora et al. (Am J Respir Crit Care Med Vol 183. pp 531-538, 2011)reported an example of the application of an RNA interferencetherapeutic to the treatment of human infectious disease and also arandomized trial of an antiviral drug in respiratory syncytial virus(RSV)-infected lung transplant recipients. Zamora et al. performed arandomized, double-blind, placebocontrolled trial in LTX recipients withRSV respiratory tract infection. Patients were permitted to receivestandard of care for RSV. Aerosolized ALN-RSV01 (0.6 mg/kg) or placebowas administered daily for 3 days. This study demonstrates that an RNAitherapeutic targeting RSV can be safely administered to LTX recipientswith RSV infection. Three daily doses of ALN-RSV01 did not result in anyexacerbation of respiratory tract symptoms or impairment of lungfunction and did not exhibit any systemic proinflammatory effects, suchas induction of cytokines or CRP. Pharmacokinetics showed only low,transient systemic exposure after inhalation, consistent withpreclinical animal data showing that ALN-RSV01, administeredintravenously or by inhalation, is rapidly cleared from the circulationthrough exonucleasemediated digestion and renal excretion. The method ofZamora et al. may be applied to the nucleic acid-targeting system of thepresent invention and an aerosolized CRISPR Cas, for example with adosage of 0.6 mg/kg, may be contemplated for the present invention.

Subjects treated for a lung disease may for example receivepharmaceutically effective amount of aerosolized AAV vector system perlung endobronchially delivered while spontaneously breathing. As such,aerosolized delivery is preferred for AAV delivery in general. Anadenovirus or an AAV particle may be used for delivery. Suitable geneconstructs, each operably linked to one or more regulatory sequences,may be cloned into the delivery vector. In this instance, the followingconstructs are provided as examples: Cbh or EF1a promoter for Cas (C2c1or C2c3), U6 or H1 promoter for guide RNA): A preferred arrangement isto use a CFTRdelta508 targeting guide, a repair template for deltaF508mutation and a codon optimized C2c1 or C2c3 enzyme, with optionally oneor more nuclear localization signal or sequence(s) (NLS(s)), e.g., two(2) NLSs. Constructs without NLS are also envisaged.

Treating Diseases of the Muscular System

The present invention also contemplates delivering the CRISPR-Cas systemdescribed herein, e.g. C2c1 or C2c3 effector protein systems, tomuscle(s).

Bortolanza et al. (Molecular Therapy vol. 19 no. 11, 2055-264 Nov. 2011)shows that systemic delivery of RNA interference expression cassettes inthe FRG1 mouse, after the onset of facioscapulohumeral musculardystrophy (FSHD), led to a dose-dependent long-term FRG1 knockdownwithout signs of toxicity. Bortolanza et al. found that a singleintravenous injection of 5×10¹² vg of rAAV6-sh1FRG1 rescues musclehistopathology and muscle function of FRG1 mice. In detail, 200 μlcontaining 2×10¹² or 5×10¹² vg of vector in physiological solution wereinjected into the tail vein using a 25-gauge Terumo syringe. The methodof Bortolanza et al. may be applied to an AAV expressing CRISPR Cas andinjected into humans at a dosage of about 2×10¹⁵ or 2×10¹⁶ vg of vector.

Dumonceaux et al. (Molecular Therapy vol. 18 no. 5, 881-887 May 2010)inhibit the myostatin pathway using the technique of RNA interferencedirected against the myostatin receptor AcvRIIb mRNA (sh-AcvRIIb). Therestoration of a quasi-dystrophin was mediated by the vectorized U7exon-skipping technique (U7-DYS). Adeno-associated vectors carryingeither the sh-AcvrIIb construct alone, the U7-DYS construct alone, or acombination of both constructs were injected in the tibialis anterior(TA) muscle of dystrophic mdx mice. The injections were performed with10¹¹ AAV viral genomes. The method of Dumonceaux et al. may be appliedto an AAV expressing CRISPR Cas and injected into humans, for example,at a dosage of about 10¹⁴ to about 10¹⁵ vg of vector.

Kinouchi et al. (Gene Therapy (2008) 15, 1126-1130) report theeffectiveness of in vivo siRNA delivery into skeletal muscles of normalor diseased mice through nanoparticle formation of chemically unmodifiedsiRNAs with atelocollagen (ATCOL). ATCOL-mediated local application ofsiRNA targeting myostatin, a negative regulator of skeletal musclegrowth, in mouse skeletal muscles or intravenously, caused a markedincrease in the muscle mass within a few weeks after application. Theseresults imply that ATCOL-mediated application of siRNAs is a powerfultool for future therapeutic use for diseases including muscular atrophy.MstsiRNAs (final concentration, 10 mM) were mixed with ATCOL (finalconcentration for local administration, 0.5%) (AteloGene, Kohken, Tokyo,Japan) according to the manufacturer's instructions. After anesthesia ofmice (20-week-old male C57BL/6) by Nembutal (25 mg/kg, i.p.), theMst-siRNA/ATCOL complex was injected into the masseter and bicepsfemoris muscles. The method of Kinouchi et al. may be applied to CRISPRCas and injected into a human, for example, at a dosage of about 500 to1000 ml of a 40 sM solution into the muscle. Hagstrom et al. (MolecularTherapy Vol. 10, No. 2, August 2004) describe an intravascular, nonviralmethodology that enables efficient and repeatable delivery of nucleicacids to muscle cells (myofibers) throughout the limb muscles ofmammals. The procedure involves the injection of naked plasmid DNA orsiRNA into a distal vein of a limb that is transiently isolated by atourniquet or blood pressure cuff. Nucleic acid delivery to myofibers isfacilitated by its rapid injection in sufficient volume to enableextravasation of the nucleic acid solution into muscle tissue. Highlevels of transgene expression in skeletal muscle were achieved in bothsmall and large animals with minimal toxicity. Evidence of siRNAdelivery to limb muscle was also obtained. For plasmid DNA intravenousinjection into a rhesus monkey, a threeway stopcock was connected to twosyringe pumps (Model PHD 2000; Harvard Instruments), each loaded with asingle syringe. Five minutes after a papaverine injection, pDNA (15.5 to25.7 mg in 40 −100 ml saline) was injected at a rate of 1.7 or 2.0 ml/s.This could be scaled up for plasmid DNA expressing CRISPR Cas of thepresent invention with an injection of about 300 to 500 mg in 800 to2000 ml saline for a human. For adenoviral vector injections into a rat,2×10⁹ infectious particles were injected in 3 ml of normal salinesolution (NSS). This could be scaled up for an adenoviral vectorexpressing CRISPR Cas of the present invention with an injection ofabout 1×10¹³ infectious particles were injected in 10 liters of NSS fora human. For siRNA, a rat was injected into the great saphenous veinwith 12.5 μg of a siRNA and a primate was injected into the greatsaphenous vein with 750 μg of a siRNA. This could be scaled up for aCRISPR Cas of the present invention, for example, with an injection ofabout 15 to about 50 mg into the great saphenous vein of a human.

See also, for example, WO2013163628 A2, Genetic Correction of MutatedGenes, published application of Duke University describes efforts tocorrect, for example, a frameshift mutation which causes a prematurestop codon and a truncated gene product that can be corrected vianuclease mediated non-homologous end joining such as those responsiblefor Duchenne Muscular Dystrophy, (“DMD”) a recessive, fatal, X-linkeddisorder that results in muscle degeneration due to mutations in thedystrophin gene. The majority of dystrophin mutations that cause DMD aredeletions of exons that disrupt the reading frame and cause prematuretranslation termination in the dystrophin gene. Dystrophin is acytoplasmic protein that provides structural stability to thedystroglycan complex of the cell membrane that is responsible forregulating muscle cell integrity and function. The dystrophin gene or“DMD gene” as used interchangeably herein is 2.2 megabases at locusXp21. The primary transcription measures about 2,400 kb with the maturemRNA being about 14 kb. 79 exons code for the protein which is over 3500amino acids. Exon 51 is frequently adjacent to frame-disruptingdeletions in DMD patients and has been targeted in clinical trials foroligonucleotide-based exon skipping. A clinical trial for the exon 51skipping compound eteplirsen recently reported a significant functionalbenefit across 48 weeks, with an average of 47% dystrophin positivefibers compared to baseline. Mutations in exon 51 are ideally suited forpermanent correction by NHEJ-based genome editing.

The methods of US Patent Publication No. 20130145487 assigned toCellectis, which relates to meganuclease variants to cleave a targetsequence from the human dystrophin gene (DMD), may also be modified tofor the nucleic acid-targeting system of the present invention.

Treating Diseases of the Skin

The present invention also contemplates delivering the CRISPR-Cas systemdescribed herein, e.g. C2c1 or C2c3 effector protein systems, to theskin.

Hickerson et al. (Molecular Therapy—Nucleic Acids (2013) 2, e129)relates to a motorized microneedle array skin delivery device fordelivering self-delivery (sd)-siRNA to human and murine skin. Theprimary challenge to translating siRNA-based skin therapeutics to theclinic is the development of effective delivery systems. Substantialeffort has been invested in a variety of skin delivery technologies withlimited success. In a clinical study in which skin was treated withsiRNA, the exquisite pain associated with the hypodermic needleinjection precluded enrollment of additional patients in the trial,highlighting the need for improved, more “patient-friendly” (i.e.,little or no pain) delivery approaches. Microneedles represent anefficient way to deliver large charged cargos including siRNAs acrossthe primary barrier, the stratum corneum, and are generally regarded asless painful than conventional hypodermic needles. Motorized “stamptype” microneedle devices, including the motorized microneedle array(MMNA) device used by Hickerson et al., have been shown to be safe inhairless mice studies and cause little or no pain as evidenced by (i)widespread use in the cosmetic industry and (ii) limited testing inwhich nearly all volunteers found use of the device to be much lesspainful than a flushot, suggesting siRNA delivery using this device willresult in much less pain than was experienced in the previous clinicaltrial using hypodermic needle injections. The MMNA device (marketed asTriple-M or Tri-M by Bomtech Electronic Co, Seoul, South Korea) wasadapted for delivery of siRNA to mouse and human skin. sd-siRNA solution(up to 300 μl of 0.1 mg/ml RNA) was introduced into the chamber of thedisposable Tri-M needle cartridge (Bomtech), which was set to a depth of0.1 mm. For treating human skin, deidentified skin (obtained immediatelyfollowing surgical procedures) was manually stretched and pinned to acork platform before treatment. All intradermal injections wereperformed using an insulin syringe with a 28-gauge 0.5-inch needle. TheMMNA device and method of Hickerson et al. could be used and/or adaptedto deliver the CRISPR Cas of the present invention, for example, at adosage of up to 300 μl of 0.1 mg/ml CRISPR Cas to the skin.

Leachman et al. (Molecular Therapy, vol. 18 no. 2, 442-446 Feb. 2010)relates to a phase 1b clinical trial for treatment of a rare skindisorder pachyonychia congenita (PC), an autosomal dominant syndromethat includes a disabling plantar keratoderma, utilizing the firstshort-interfering RNA (siRNA)-based therapeutic for skin. This siRNA,called TD101, specifically and potently targets the keratin 6a (K6a)N171K mutant mRNA without affecting wild-type K6a mRNA.

Zheng et al. (PNAS, Jul. 24, 2012, vol. 109, no. 30, 11975-11980) showthat spherical nucleic acid nanoparticle conjugates (SNA-NCs), goldcores surrounded by a dense shell of highly oriented, covalentlyimmobilized siRNA, freely penetrate almost 100% of keratinocytes invitro, mouse skin, and human epidermis within hours after application.Zheng et al. demonstrated that a single application of 25 nM epidermalgrowth factor receptor (EGFR) SNA-NCs for 60 h demonstrate effectivegene knockdown in human skin. A similar dosage may be contemplated forCRISPR Cas immobilized in SNA-NCs for administration to the skin.

Cancer

In some embodiments, the treatment, prophylaxis or diagnosis of canceris provided. The target is preferably one or more of the FAS, BID,CTLA4, PDCD1, CBLB, PTPN6, TRAC or TRBC genes. The cancer may be one ormore of lymphoma, chronic lymphocytic leukemia (CLL), B cell acutelymphocytic leukemia (B-ALL), acute lymphoblastic leukemia, acutemyeloid leukemia, non-Hodgkin's lymphoma (NHL), diffuse large celllymphoma (DLCL), multiple myeloma, renal cell carcinoma (RCC),neuroblastoma, colorectal cancer, breast cancer, ovarian cancer,melanoma, sarcoma, prostate cancer, lung cancer, esophageal cancer,hepatocellular carcinoma, pancreatic cancer, astrocytoma, mesothelioma,head and neck cancer, and medulloblastoma. This may be implemented withengineered chimeric antigen receptor (CAR) T cell. This is described inWO2015161276, the disclosure of which is hereby incorporated byreference and described herein below.

Target genes suitable for the treatment or prophylaxis of cancer mayinclude, in some embodiments, those described in WO2015048577 thedisclosure of which is hereby incorporated by reference.

Usher Syndrome or Retinitis Pigmentosa-39

In some embodiments, the treatment, prophylaxis or diagnosis of UsherSyndrome or retinitis pigmentosa-39 is provided. The target ispreferably the USH2A gene. In some embodiments, correction of a Gdeletion at position 2299 (2299delG) is provided. This is described inWO2015134812A1, the disclosure of which is hereby incorporated byreference.

Cystic Fibrosis (CF)

In some embodiments, the treatment, prophylaxis or diagnosis of cysticfibrosis is provided. The target is preferably the SCNN1A or the CFTRgene. This is described in WO2015157070, the disclosure of which ishereby incorporated by reference.

Schwank et al. (Cell Stem Cell, 13:653-58, 2013) used CRISPR-Cas9 tocorrect a defect associated with cystic fibrosis in human stem cells.The team's target was the gene for an ion channel, cystic fibrosistransmembrane conductor receptor (CFTR). A deletion in CFTR causes theprotein to misfold in cystic fibrosis patients. Using culturedintestinal stem cells developed from cell samples from two children withcystic fibrosis, Schwank et al. were able to correct the defect usingCRISPR along with a donor plasmid containing the reparative sequence tobe inserted. The researchers then grew the cells into intestinal“organoids,” or miniature guts, and showed that they functionednormally. In this case, about half of clonal organoids underwent theproper genetic correction.

HIV and AIDS

In some embodiments, the treatment, prophylaxis or diagnosis of HIV andAIDS is provided. The target is preferably the CCR5 gene in HIV. This isdescribed in WO2015148670A1, the disclosure of which is herebyincorporated by reference.

Beta Thalassaemia

In some embodiments, the treatment, prophylaxis or diagnosis of BetaThalassaemia is provided. The target is preferably the BCL11A gene. Thisis described in WO2015148860, the disclosure of which is herebyincorporated by reference.

Sickle Cell Disease (SCD)

In some embodiments, the treatment, prophylaxis or diagnosis of SickleCell Disease (SCD) is provided. The target is preferably the HBB orBCL11A gene. This is described in WO2015148863, the disclosure of whichis hereby incorporated by reference.

Heroes Simplex Virus 1 and 2

In some embodiments, the treatment, prophylaxis or diagnosis of HSV-1(Herpes Simplex Virus 1) is provided. The target is preferably the UL19,UL30, UL48 or UL50 gene in HSV-1. This is described in WO2015153789, thedisclosure of which is hereby incorporated by reference.

In other embodiments, the treatment, prophylaxis or diagnosis of HSV-2(Herpes Simplex Virus 2) is provided. The target is preferably the UL19,UL30, UL48 or UL50 gene in HSV-2. This is described in WO2015153791, thedisclosure of which is hereby incorporated by reference.

In some embodiments, the treatment, prophylaxis or diagnosis of PrimaryOpen Angle Glaucoma (POAG) is provided. The target is preferably theMYOC gene. This is described in WO2015153780, the disclosure of which ishereby incorporated by reference.

Adoptive Cell Therapies

The present invention also contemplates use of the CRISPR-Cas systemdescribed herein, e.g. C2c1 or C2c3 effector protein systems, to modifycells for adoptive therapies. Aspects of the invention accordinglyinvolve the adoptive transfer of immune system cells, such as T cells,specific for selected antigens, such as tumor associated antigens (seeMaus et al., 2014, Adoptive Immunotherapy for Cancer or Viruses, AnnualReview of Immunology, Vol. 32: 189-225; Rosenberg and Restifo, 2015,Adoptive cell transfer as personalized immunotherapy for human cancer,Science Vol. 348 no. 6230 pp. 62-68; and, Restifo et al., 2015, Adoptiveimmunotherapy for cancer: harnessing the T cell response. Nat. Rev.Immunol. 12(4): 269-281; and Jenson and Riddell, 2014, Design andimplementation of adoptive therapy with chimeric antigenreceptor-modified T cells. Immunol Rev. 257(1): 127-144). Variousstrategies may for example be employed to genetically modify T cells byaltering the specificity of the T cell receptor (TCR) for example byintroducing new TCR α and β chains with selected peptide specificity(see U.S. Pat. No. 8,697,854; PCT Patent Publications: WO2003020763,WO2004033685, WO2004044004, WO2005114215, WO2006000830, WO2008038002,WO2008039818, WO2004074322, WO2005113595, WO2006125962, WO2013166321,WO2013039889, WO2014018863, WO2014083173; U.S. Pat. No. 8,088,379).

As an alternative to, or addition to, TCR modifications, chimericantigen receptors (CARs) may be used in order to generateimmunoresponsive cells, such as T cells, specific for selected targets,such as malignant cells, with a wide variety of receptor chimeraconstructs having been described (see U.S. Pat. Nos. 5,843,728;5,851,828; 5,912,170; 6,004,811; 6,284,240; 6,392,013; 6,410,014;6,753,162; 8,211,422; and, PCT Publication WO9215322). Alternative CARconstructs may be characterized as belonging to successive generations.First-generation CARs typically consist of a single-chain variablefragment of an antibody specific for an antigen, for example comprisinga VL linked to a VH of a specific antibody, linked by a flexible linker,for example by a CD8α hinge domain and a CD8α transmembrane domain, tothe transmembrane and intracellular signaling domains of either CD3ζ orFcRγ (scFv-CD3ζ or scFv-FcRγ; see U.S. Pat. Nos. 7,741,465; 5,912,172;5,906,936). Second-generation CARs incorporate the intracellular domainsof one or more costimulatory molecules, such as CD28, OX40 (CD134), or4-1BB (CD137) within the endodomain (for examplescFv-CD28/OX40/4-1BB-CD3ζ; see U.S. Pat. Nos. 8,911,993; 8,916,381;8,975,071; 9,101,584; 9,102,760; 9,102,761). Third-generation CARsinclude a combination of costimulatory endodomains, such a CD3ζ-chain,CD97, GDI la-CD18, CD2, ICOS, CD27, CD154, CDS, OX40, 4-1BB, or CD28signaling domains (for example scFv-CD28-4-1BB-CD3ζ orscFv-CD28-OX40-CD3ζ; see U.S. Pat. Nos. 8,906,682; 8,399,645; 5,686,281;PCT Publication No. WO2014134165; PCT Publication No. WO2012079000).Alternatively, costimulation may be orchestrated by expressing CARs inantigen-specific T cells, chosen so as to be activated and expandedfollowing engagement of their native αβTCR, for example by antigen onprofessional antigen-presenting cells, with attendant costimulation. Inaddition, additional engineered receptors may be provided on theimmunoresponsive cells, for example to improve targeting of a T-cellattack and/or minimize side effects.

Alternative techniques may be used to transform target immunoresponsivecells, such as protoplast fusion, lipofection, transfection orelectroporation. A wide variety of vectors may be used, such asretroviral vectors, lentiviral vectors, adenoviral vectors,adeno-associated viral vectors, plasmids or transposons, such as aSleeping Beauty transposon (see U.S. Pat. Nos. 6,489,458; 7,148,203;7,160,682; 7,985,739; 8,227,432), may be used to introduce CARs, forexample using 2nd generation antigen-specific CARs signaling throughCD3ζ and either CD28 or CD137. Viral vectors may for example includevectors based on HIV, SV40, EBV, HSV or BPV.

Cells that are targeted for transformation may for example include Tcells, Natural Killer (NK) cells, cytotoxic T lymphocytes (CTL),regulatory T cells, human embryonic stem cells, tumor-infiltratinglymphocytes (TIL) or a pluripotent stem cell from which lymphoid cellsmay be differentiated. T cells expressing a desired CAR may for examplebe selected through co-culture with 7-irradiated activating andpropagating cells (AaPC), which co-express the cancer antigen andco-stimulatory molecules. The engineered CAR T-cells may be expanded,for example by co-culture on AaPC in presence of soluble factors, suchas IL-2 and IL-21. This expansion may for example be carried out so asto provide memory CAR+ T cells (which may for example be assayed bynon-enzymatic digital array and/or multi-panel flow cytometry). In thisway, CAR T cells may be provided that have specific cytotoxic activityagainst antigen-bearing tumors (optionally in conjunction withproduction of desired chemokines such as interferon-y). CAR T cells ofthis kind may for example be used in animal models, for example tothreat tumor xenografts.

Approaches such as the foregoing may be adapted to provide methods oftreating and/or increasing survival of a subject having a disease, suchas a neoplasia, for example by administering an effective amount of animmunoresponsive cell comprising an antigen recognizing receptor thatbinds a selected antigen, wherein the binding activates theimmunoresponsive cell, thereby treating or preventing the disease (suchas a neoplasia, a pathogen infection, an autoimmune disorder, or anallogeneic transplant reaction). Dosing in CAR T cell therapies may forexample involve administration of from 106 to 109 cells/kg, with orwithout a course of lymphodepletion, for example with cyclophosphamide.

In one embodiment, the treatment can be administrated into patientsundergoing an immunosuppressive treatment. The cells or population ofcells, may be made resistant to at least one immunosuppressive agent dueto the inactivation of a gene encoding a receptor for suchimmunosuppressive agent. Not being bound by a theory, theimmunosuppressive treatment should help the selection and expansion ofthe immunoresponsive or T cells according to the invention within thepatient.

The administration of the cells or population of cells according to thepresent invention may be carried out in any convenient manner, includingby aerosol inhalation, injection, ingestion, transfusion, implantationor transplantation. The cells or population of cells may be administeredto a patient subcutaneously, intradermally, intratumorally,intranodally, intramedullary, intramuscularly, by intravenous orintralymphatic injection, or intraperitoneally. In one embodiment, thecell compositions of the present invention are preferably administeredby intravenous injection.

The administration of the cells or population of cells can consist ofthe administration of 10⁴-10⁹ cells per kg body weight, preferably 10⁵to 10⁶ cells/kg body weight including all integer values of cell numberswithin those ranges. Dosing in CAR T cell therapies may for exampleinvolve administration of from 10⁶ to 10⁹ cells/kg, with or without acourse of lymphodepletion, for example with cyclophosphamide. The cellsor population of cells can be administrated in one or more doses. Inanother embodiment, the effective amount of cells are administrated as asingle dose. In another embodiment, the effective amount of cells areadministrated as more than one dose over a period time. Timing ofadministration is within the judgment of managing physician and dependson the clinical condition of the patient. The cells or population ofcells may be obtained from any source, such as a blood bank or a donor.While individual needs vary, determination of optimal ranges ofeffective amounts of a given cell type for a particular disease orconditions are within the skill of one in the art. An effective amountmeans an amount which provides a therapeutic or prophylactic benefit.The dosage administrated will be dependent upon the age, health andweight of the recipient, kind of concurrent treatment, if any, frequencyof treatment and the nature of the effect desired.

In another embodiment, the effective amount of cells or compositioncomprising those cells are administrated parenterally. Theadministration can be an intravenous administration. The administrationcan be directly done by injection within a tumor.

To guard against possible adverse reactions, engineered immunoresponsivecells may be equipped with a transgenic safety switch, in the form of atransgene that renders the cells vulnerable to exposure to a specificsignal. For example, the herpes simplex viral thymidine kinase (TK) genemay be used in this way, for example by introduction into allogeneic Tlymphocytes used as donor lymphocyte infusions following stem celltransplantation (Greco, et al., Improving the safety of cell therapywith the TK-suicide gene. Front. Pharmacol. 2015; 6: 95). In such cells,administration of a nucleoside prodrug such as ganciclovir or acyclovircauses cell death. Alternative safety switch constructs includeinducible caspase 9, for example triggered by administration of asmall-molecule dimerizer that brings together two nonfunctional icasp9molecules to form the active enzyme. A wide variety of alternativeapproaches to implementing cellular proliferation controls have beendescribed (see U.S. Patent Publication No. 20130071414; PCT PatentPublication WO2011146862; PCT Patent Publication WO2014011987; PCTPatent Publication WO2013040371; Zhou et al. BLOOD, 2014,123/25:3895-3905; Di Stasi et al., The New England Journal of Medicine2011; 365:1673-1683; Sadelain M, The New England Journal of Medicine2011; 365:1735-173; Ramos et al., Stem Cells 28(6):1107-15 (2010)).

In a further refinement of adoptive therapies, genome editing with aCRISPR-Cas system as described herein may be used to tailorimmunoresponsive cells to alternative implementations, for exampleproviding edited CAR T cells (see Poirot et al., 2015, Multiplex genomeedited T-cell manufacturing platform for “off-the-shelf” adoptive T-cellimmunotherapies, Cancer Res 75 (18): 3853). For example,immunoresponsive cells may be edited to delete expression of some or allof the class of HLA type II and/or type I molecules, or to knockoutselected genes that may inhibit the desired immune response, such as thePD1 gene.

Cells may be edited using any CRISPR system and method of use thereof asdescribed herein. CRISPR systems may be delivered to an immune cell byany method described herein. In preferred embodiments, cells are editedex vivo and transferred to a subject in need thereof. Immunoresponsivecells, CAR T cells or any cells used for adoptive cell transfer may beedited. Editing may be performed to eliminate potential alloreactiveT-cell receptors (TCR), disrupt the target of a chemotherapeutic agent,block an immune checkpoint, activate a T cell, and/or increase thedifferentiation and/or proliferation of functionally exhausted ordysfunctional CD8+ T-cells (see PCT Patent Publications: WO2013176915,WO2014059173, WO2014172606, WO2014184744, and WO2014191128). Editing mayresult in inactivation of a gene.

By inactivating a gene it is intended that the gene of interest is notexpressed in a functional protein form. In a particular embodiment, theCRISPR system specifically catalyzes cleavage in one targeted genethereby inactivating said targeted gene. The nucleic acid strand breakscaused are commonly repaired through the distinct mechanisms ofhomologous recombination or non-homologous end joining (NHEJ). However,NHEJ is an imperfect repair process that often results in changes to theDNA sequence at the site of the cleavage. Repair via non-homologous endjoining (NHEJ) often results in small insertions or deletions (Indel)and can be used for the creation of specific gene knockouts. Cells inwhich a cleavage induced mutagenesis event has occurred can beidentified and/or selected by well-known methods in the art.

T cell receptors (TCR) are cell surface receptors that participate inthe activation of T cells in response to the presentation of antigen.The TCR is generally made from two chains, α and β, which assemble toform a heterodimer and associates with the CD3-transducing subunits toform the T cell receptor complex present on the cell surface. Each α andβ chain of the TCR consists of an immunoglobulin-like N-terminalvariable (V) and constant (C) region, a hydrophobic transmembranedomain, and a short cytoplasmic region. As for immunoglobulin molecules,the variable region of the a and p chains are generated by V(D)Jrecombination, creating a large diversity of antigen specificitieswithin the population of T cells. However, in contrast toimmunoglobulins that recognize intact antigen, T cells are activated byprocessed peptide fragments in association with an MHC molecule,introducing an extra dimension to antigen recognition by T cells, knownas MHC restriction. Recognition of MHC disparities between the donor andrecipient through the T cell receptor leads to T cell proliferation andthe potential development of graft versus host disease (GVHD). Theinactivation of TCRα or TCRβ can result in the elimination of the TCRfrom the surface of T cells preventing recognition of alloantigen andthus GVHD. However, TCR disruption generally results in the eliminationof the CD3 signaling component and alters the means of further T cellexpansion.

Allogeneic cells are rapidly rejected by the host immune system. It hasbeen demonstrated that, allogeneic leukocytes present in non-irradiatedblood products will persist for no more than 5 to 6 days (Boni, Muranskiet al. 2008 Blood 1; 112(12):4746-54). Thus, to prevent rejection ofallogeneic cells, the host's immune system usually has to be suppressedto some extent. However, in the case of adoptive cell transfer the useof immunosuppressive drugs also have a detrimental effect on theintroduced therapeutic T cells. Therefore, to effectively use anadoptive immunotherapy approach in these conditions, the introducedcells would need to be resistant to the immunosuppressive treatment.Thus, in a particular embodiment, the present invention furthercomprises a step of modifying T cells to make them resistant to animmunosuppressive agent, preferably by inactivating at least one geneencoding a target for an immunosuppressive agent. An immunosuppressiveagent is an agent that suppresses immune function by one of severalmechanisms of action. An immunosuppressive agent can be, but is notlimited to a calcineurin inhibitor, a target of rapamycin, aninterleukin-2 receptor a-chain blocker, an inhibitor of inosinemonophosphate dehydrogenase, an inhibitor of dihydrofolic acidreductase, a corticosteroid or an immunosuppressive antimetabolite. Thepresent invention allows conferring immunosuppressive resistance to Tcells for immunotherapy by inactivating the target of theimmunosuppressive agent in T cells. As non-limiting examples, targetsfor an immunosuppressive agent can be a receptor for animmunosuppressive agent such as: CD52, glucocorticoid receptor (GR), aFKBP family gene member and a cyclophilin family gene member.

Immune checkpoints are inhibitory pathways that slow down or stop immunereactions and prevent excessive tissue damage from uncontrolled activityof immune cells. In certain embodiments, the immune checkpoint targetedis the programmed death-1 (PD-1 or CD279) gene (PDCD1). In otherembodiments, the immune checkpoint targeted is cytotoxicT-lymphocyte-associated antigen (CTLA-4). In additional embodiments, theimmune checkpoint targeted is another member of the CD28 and CTLA4 Igsuperfamily such as BTLA, LAG3, ICOS, PDL1 or KIR. In further additionalembodiments, the immune checkpoint targeted is a member of the TNFRsuperfamily such as CD40, OX40, CD137, GITR, CD27 or TIM-3.

Additional immune checkpoints include Src homology 2 domain-containingprotein tyrosine phosphatase 1 (SHP-1) (Watson H A, et al., SHP-1: thenext checkpoint target for cancer immunotherapy? Biochem Soc Trans. 2016Apr. 15; 44(2):356-62). SHP-1 is a widely expressed inhibitory proteintyrosine phosphatase (PTP). In T-cells, it is a negative regulator ofantigen-dependent activation and proliferation. It is a cytosolicprotein, and therefore not amenable to antibody-mediated therapies, butits role in activation and proliferation makes it an attractive targetfor genetic manipulation in adoptive transfer strategies, such aschimeric antigen receptor (CAR) T cells. Immune checkpoints may alsoinclude T cell immunoreceptor with Ig and ITIM domains(TIGIT/Vstm3/WUCAM/VSIG9) and VISTA (Le Mercier I, et al., (2015) BeyondCTLA-4 and PD-1, the generation Z of negative checkpoint regulators.Front. Immunol. 6:418).

WO2014172606 relates to the use of MT1 and/or MT1 inhibitors to increaseproliferation and/or activity of exhausted CD8+ T-cells and to decreaseCD8+ T-cell exhaustion (e.g., decrease functionally exhausted orunresponsive CD8+ immune cells). In certain embodiments,metallothioneins are targeted by gene editing in adoptively transferredT cells.

In certain embodiments, targets of gene editing may be at least onetargeted locus involved in the expression of an immune checkpointprotein. Such targets may include, but are not limited to CTLA4, PPP2CA,PPP2CB, PTPN6, PTPN22, PDCD1, ICOS (CD278), PDL1, KIR, LAG3, HAVCR2,BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244 (2B4),TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS,TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA,IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1,BATF, VISTA, GUCYIA2, GUCYIA3, GUCYIB2, GUCYIB3, MT1, MT2, CD40, OX40,CD137, GITR, CD27, SHP-1 or TIM-3. In preferred embodiments, the genelocus involved in the expression of PD-1 or CTLA-4 genes is targeted. Inother preferred embodiments, combinations of genes are targeted, such asbut not limited to PD-1 and TIGIT.

In other embodiments, at least two genes are edited. Pairs of genes mayinclude, but are not limited to PD1 and TCRα, PD1 and TCRβ, CTLA-4 andTCRα, CTLA-4 and TCRβ, LAG3 and TCRα, LAG3 and TCRβ, Tim3 and TCRα, Tim3and TCRβ, BTLA and TCRα, BTLA and TCRβ, BY55 and TCRα, BY55 and TCRβ,TIGIT and TCRα, TIGIT and TCRβ, B7H5 and TCRα, B7H5 and TCRβ, LAIR1 andTCRα, LAIR1 and TCRβ, SIGLEC1O and TCRα, SIGLEC10 and TCRβ, 2B4 andTCRα, 2B4 and TCRβ.

Whether prior to or after genetic modification of the T cells, the Tcells can be activated and expanded generally using methods asdescribed, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055;6,905,680; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,232,566;7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and 7,572,631. Tcells can be expanded in vitro or in vivo.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of immunology, biochemistry,chemistry, molecular biology, microbiology, cell biology, genomics andrecombinant DNA, which are within the skill of the art. See MOLECULARCLONING: A LABORATORY MANUAL, 2nd edition (1989) (Sambrook, Fritsch andManiatis); MOLECULAR CLONING: A LABORATORY MANUAL, 4th edition (2012)(Green and Sambrook); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (1987) (F.M. Ausubel, et al. eds.); the series METHODS IN ENZYMOLOGY (AcademicPress, Inc.); PCR 2: A PRACTICAL APPROACH (1995) (M. J. MacPherson, B.D. Hames and G. R. Taylor eds.); ANTIBODIES, A LABORATORY MANUAL (1988)(Harlow and Lane, eds.); ANTIBODIES A LABORATORY MANUAL, 2nd edition(2013) (E. A. Greenfield ed.); and ANIMAL CELL CULTURE (1987) (R.I.Freshney, ed.).

The practice of the present invention employs, unless otherwiseindicated, conventional techniques for generation of geneticallymodified mice. See Marten H. Hofker and Jan van Deursen, TRANSGENICMOUSE METHODS AND PROTOCOLS, 2nd edition (2011).

Gene Drives

The present invention also contemplates use of the CRISPR-Cas systemdescribed herein, e.g. C2c1 or C2c3 effector protein systems, to provideRNA-guided gene drives, for example in systems analogous to gene drivesdescribed in PCT Patent Publication WO 2015/105928. Systems of this kindmay for example provide methods for altering eukaryotic germline cells,by introducing into the germline cell a nucleic acid sequence encodingan RNA-guided DNA nuclease and one or more guide RNAs. The guide RNAsmay be designed to be complementary to one or more target locations ongenomic DNA of the germline cell. The nucleic acid sequence encoding theRNA guided DNA nuclease and the nucleic acid sequence encoding the guideRNAs may be provided on constructs between flanking sequences, withpromoters arranged such that the germline cell may express the RNAguided DNA nuclease and the guide RNAs, together with any desiredcargo-encoding sequences that are also situated between the flankingsequences. The flanking sequences will typically include a sequencewhich is identical to a corresponding sequence on a selected targetchromosome, so that the flanking sequences work with the componentsencoded by the construct to facilitate insertion of the foreign nucleicacid construct sequences into genomic DNA at a target cut site bymechanisms such as homologous recombination, to render the germline cellhomozygous for the foreign nucleic acid sequence. In this way,gene-drive systems are capable of introgressing desired cargo genesthroughout a breeding population (Gantz et al., 2015, Highly efficientCas9-mediated gene drive for population modification of the malariavector mosquito Anopheles stephensi, PNAS 2015, published ahead of printNov. 23, 2015, doi:10.1073/pnas.1521077112; Esvelt et al., 2014,Concerning RNA-guided gene drives for the alteration of wild populationseLife 2014; 3:e03401). In select embodiments, target sequences may beselected which have few potential off-target sites in a genome.Targeting multiple sites within a target locus, using multiple guideRNAs, may increase the cutting frequency and hinder the evolution ofdrive resistant alleles. Truncated guide RNAs may reduce off-targetcutting. Paired nickases may be used instead of a single nuclease, tofurther increase specificity. Gene drive constructs may include cargosequences encoding transcriptional regulators, for example to activatehomologous recombination genes and/or repress non-homologousend-joining. Target sites may be chosen within an essential gene, sothat non-homologous end-joining events may cause lethality rather thancreating a drive-resistant allele. The gene drive constructs can beengineered to function in a range of hosts at a range of temperatures(Cho et al. 2013, Rapid and Tunable Control of Protein Stability inCaenorhabditis elegans Using a Small Molecule, PLoS ONE 8(8): e72393.doi:10.1371/journal.pone.0072393).

Xenotransplantation

The present invention also contemplates use of the CRISPR-Cas systemdescribed herein, e.g. C2c1 or C2c3 effector protein systems, to provideRNA-guided DNA nucleases adapted to be used to provide modified tissuesfor transplantation. For example, RNA-guided DNA nucleases may be usedto knockout, knockdown or disrupt selected genes in an animal, such as atransgenic pig (such as the human heme oxygenase-1 transgenic pig line),for example by disrupting expression of genes that encode epitopesrecognized by the human immune system, i.e. xenoantigen genes. Candidateporcine genes for disruption may for example includea(1,3)-galactosyltransferase and cytidinemonophosphate-N-acetylneuraminic acid hydroxylase genes (see PCT PatentPublication WO 2014/066505). In addition, genes encoding endogenousretroviruses may be disrupted, for example the genes encoding allporcine endogenous retroviruses (see Yang et al., 2015, Genome-wideinactivation of porcine endogenous retroviruses (PERVs), Science 27 Nov.2015: Vol. 350 no. 6264 pp. 1101-1104). In addition, RNA-guided DNAnucleases may be used to target a site for integration of additionalgenes in xenotransplant donor animals, such as a human CD55 gene toimprove protection against hyperacute rejection.

General Gene Therapy Considerations

Examples of disease-associated genes and polynucleotides and diseasespecific information is available from McKusick-Nathans Institute ofGenetic Medicine, Johns Hopkins University (Baltimore, Md.) and NationalCenter for Biotechnology Information, National Library of Medicine(Bethesda, Md.), available on the World Wide Web.

Mutations in these genes and pathways can result in production ofimproper proteins or proteins in improper amounts which affect function.Further examples of genes, diseases and proteins are hereby incorporatedby reference from U.S. Provisional application 61/736,527 filed Dec. 12,2012. Such genes, proteins and pathways may be the target polynucleotideof a CRISPR complex of the present invention. Examples ofdisease-associated genes and polynucleotides are listed in Tables 10 and11. Examples of signaling biochemical pathway-associated genes andpolynucleotides are listed in Table 12.

TABLE 10 DISEASE/DISORDERS GENE(S) Neoplasia PTEN; ATM; ATR; EGFR;ERBB2; ERBB3; ERBB4; Notch1; Notch2; Notch3; Notch4; AKT; AKT2; AKT3;HIF; HIF1a; HIF3a; Met; HRG; Bcl2; PPAR alpha; PPAR gamma; WT1 (WilmsTumor); FGF Receptor Family members (5 members: 1, 2, 3, 4, 5); CDKN2a;APC; RB (retinoblastoma); MEN1; VHL; BRCA1; BRCA2; AR (AndrogenReceptor); TSG101; IGF; IGF Receptor; Igf1 (4 variants); Igf2 (3variants); Igf 1 Receptor; Igf 2 Receptor; Bax; Bcl2; caspases family (9members: 1, 2, 3, 4, 6, 7, 8, 9, 12); Kras; Apc Age-related MacularAbcr; Ccl2; Cc2; cp (ceruloplasmin); Timp3; cathepsinD; DegenerationVldlr; Ccr2 Schizophrenia Neuregulin1 (Nrg1); Erb4 (receptor forNeuregulin); Complexin1 (Cplx1); Tph1 Tryptophan hydroxylase; Tph2Tryptophan hydroxylase 2; Neurexin 1; GSK3; GSK3a; GSK3b Disorders 5-HTT(Slc6a4); COMT; DRD (Drd1a); SLC6A3; DAOA; DTNBP1; Dao (Dao1)Trinucleotide HTT (Huntington's Dx); SBMA/SMAX1/AR (Kennedy's RepeatDisorders Dx); FXN/X25 (Friedrich's Ataxia); ATX3 (Machado- Joseph'sDx); ATXN1 and ATXN2 (spinocerebellar ataxias); DMPK (myotonicdystrophy); Atrophin-1 and Atn1 (DRPLA Dx); CBP (Creb-BP - globalinstability); VLDLR (Alzheimer's); Atxn7; Atxn10 Fragile X SyndromeFMR2; FXR1; FXR2; mGLUR5 Secretase Related APH-1 (alpha and beta);Presenilin (Psen1); nicastrin Disorders (Ncstn); PEN-2 Others Nos1;Parp1; Nat1; Nat2 Prion - related disorders Prp ALS SOD1; ALS2; STEX;FUS; TARDBP; VEGF (VEGF-a; VEGF-b; VEGF-c) Drug addiction Prkce(alcohol); Drd2; Drd4; ABAT (alcohol); GRIA2; Grm5; Grin1; Htr1b;Grin2a; Drd3; Pdyn; Gria1 (alcohol) Autism Mecp2; BZRAP1; MDGA2; Sema5A;Neurexin 1; Fragile X (FMR2 (AFF2); FXR1; FXR2; Mglur5) Alzheimer'sDisease E1; CHIP; UCH; UBB; Tau; LRP; PICALM; Clusterin; PS1; SORL1;CR1; Vldlr; Uba1; Uba3; CHIP28 (Aqp1, Aquaporin 1); Uchl1; Uchl3; APPInflammation IL-10; IL-1 (IL-1a; IL-1b); IL-13; IL-17 (IL-17a (CTLA8);IL- 17b; IL-17c; IL-17d; IL-17f); II-23; Cx3cr1; ptpn22; TNFa;NOD2/CARD15 for IBD; IL-6; IL-12 (IL-12a; IL-12b); CTLA4; Cx3cl1Parkinson's Disease x-Synuclein; DJ-1; LRRK2; Parkin; PINK1

TABLE 11 Blood and Anemia (CDAN1, CDA1, RPS19, DBA, PKLR, PK1, NT5C3,UMPH1, coagulation diseases PSN1, RHAG, RH50A, NRAMP2, SPTB, ALAS2,ANH1, ASB, and disorders ABCB7, ABC7, ASAT); Bare lymphocyte syndrome(TAPBP, TPSN, TAP2, ABCB3, PSF2, RING11, MHC2TA, C2TA, RFX5, RFXAP,RFX5), Bleeding disorders (TBXA2R, P2RX1, P2X1); Factor H and factorH-like 1 (HF1, CFH, HUS); Factor V and factor VIII (MCFD2); Factor VIIdeficiency (F7); Factor X deficiency (F10); Factor XI deficiency (F11);Factor XII deficiency (F12, HAF); Factor XIIIA deficiency (F13A1, F13A);Factor XIIIB deficiency (F13B); Fanconi anemia (FANCA, FACA, FA1, FA,FAA, FAAP95, FAAP90, FLJ34064, FANCB, FANCC, FACC, BRCA2, FANCD1,FANCD2, FANCD, FACD, FAD, FANCE, FACE, FANCF, XRCC9, FANCG, BRIP1,BACH1, FANCJ, PHF9, FANCL, FANCM, KIAA1596); Hemophagocyticlymphohistiocytosis disorders (PRF1, HPLH2, UNC13D, MUNC13-4, HPLH3,HLH3, FHL3); Hemophilia A (F8, F8C, HEMA); Hemophilia B (F9, HEMB),Hemorrhagic disorders (PI, ATT, F5); Leukocyde deficiencies anddisorders (ITGB2, CD18, LCAMB, LAD, EIF2B1, EIF2BA, EIF2B2, EIF2B3,EIF2B5, LVWM, CACH, CLE, EIF2B4); Sickle cell anemia (HBB); Thalassemia(HBA2, HBB, HBD, LCRB, HBA1). Cell dysregulation B-cell non-Hodgkinlymphoma (BCL7A, BCL7); Leukemia (TAL1 and oncology TCL5, SCL, TAL2,FLT3, NBS1, NBS, ZNFN1A1, IK1, LYF1, diseases and disorders HOXD4,HOX4B, BCR, CML, PHL, ALL, ARNT, KRAS2, RASK2, GMPS, AF10, ARHGEF12,LARG, KIAA0382, CALM, CLTH, CEBPA, CEBP, CHIC2, BTL, FLT3, KIT, PBT,LPP, NPM1, NUP214, D9S46E, CAN, CAIN, RUNX1, CBFA2, AML1, WHSC1L1, NSD3,FLT3, AF1Q, NPM1, NUMA1, ZNF145, PLZF, PML, MYL, STAT5B, AF10, CALM,CLTH, ARL11, ARLTS1, P2RX7, P2X7, BCR, CML, PHL, ALL, GRAF, NF1, VRNF,WSS, NFNS, PTPN11, PTP2C, SHP2, NS1, BCL2, CCND1, PRAD1, BCL1, TCRA,GATA1, GF1, ERYF1, NFE1, ABL1, NQO1, DIA4, NMOR1, NUP214, D9S46E, CAN,CAIN). Inflammation and AIDS (KIR3DL1, NKAT3, NKB1, AMB11, KIR3DS1,IFNG, CXCL12, immune related SDF1); Autoimmune lymphoproliferativesyndrome (TNFRSF6, APT1, diseases and disorders FAS, CD95, ALPS1A);Combined immunodeficiency, (IL2RG, SCIDX1, SCIDX, IMD4); HIV-1 (CCL5,SCYA5, D17S136E, TCP228), HIV susceptibility or infection (IL10, CSIF,CMKBR2, CCR2, CMKBR5, CCCKR5 (CCR5)); Immunodeficiencies (CD3E, CD3G,AICDA, AID, HIGM2, TNFRSF5, CD40, UNG, DGU, HIGM4, TNFSF5, CD40LG,HIGM1, IGM, FOXP3, IPEX, AIID, XPID, PIDX, TNFRSF14B, TACI);Inflammation (IL-10, IL-1 (IL-1a, IL-1b), IL-13, IL-17 (IL-17a (CTLA8),IL-17b, IL-17c, IL-17d, IL-17f), II-23, Cx3cr1, ptpn22, TNFa,NOD2/CARD15 for IBD, IL-6, IL-12 (IL-12a, IL-12b), CTLA4, Cx3cl1);Severe combined immunodeficiencies (SCIDs)(JAK3, JAKL, DCLRE1C, ARTEMIS,SCIDA, RAG1, RAG2, ADA, PTPRC, CD45, LCA, IL7R, CD3D, T3D, IL2RG,SCIDX1, SCIDX, IMD4). Metabolic, liver, Amyloid neuropathy (TTR, PALB);Amyloidosis (APOA1, APP, AAA, kidney and protein CVAP, AD1, GSN, FGA,LYZ, TTR, PALB); Cirrhosis (KRT18, KRT8, diseases and disorders CIRH1A,NAIC, TEX292, KIAA1988); Cystic fibrosis (CFTR, ABCC7, CF, MRP7);Glycogen storage diseases (SLC2A2, GLUT2, G6PC, G6PT, G6PT1, GAA, LAMP2,LAMPB, AGL, GDE, GBE1, GYS2, PYGL, PFKM); Hepatic adenoma, 142330 (TCF1,HNF1A, MODY3), Hepatic failure, early onset, and neurologic disorder(SCOD1, SCO1), Hepatic lipase deficiency (LIPC), Hepatoblastoma, cancerand carcinomas (CTNNB1, PDGFRL, PDGRL, PRLTS, AXIN1, AXIN, CTNNB1, TP53,P53, LFS1, IGF2R, MPRI, MET, CASP8, MCH5; Medullary cystic kidneydisease (UMOD, HNFJ, FJHN, MCKD2, ADMCKD2); Phenylketonuria (PAH, PKU1,QDPR, DHPR, PTS); Polycystic kidney and hepatic disease (FCYT, PKHD1,ARPKD, PKD1, PKD2, PKD4, PKDTS, PRKCSH, G19P1, PCLD, SEC63).Muscular/Skeletal Becker muscular dystrophy (DMD, BMD, MYF6), DuchenneMuscular diseases and disorders Dystrophy (DMD, BMD); Emery-Dreifussmuscular dystrophy (LMNA, LMN1, EMD2, FPLD, CMD1A, HGPS, LGMD1B, LMNA,LMN1, EMD2, FPLD, CMD1A); Facioscapulohumeral muscular dystrophy(FSHMD1A, FSHD1A); Muscular dystrophy (FKRP, MDC1C, LGMD2I, LAMA2, LAMM,LARGE, KIAA0609, MDC1D, FCMD, TTID, MYOT, CAPN3, CANP3, DYSF, LGMD2B,SGCG, LGMD2C, DMDA1, SCG3, SGCA, ADL, DAG2, LGMD2D, DMDA2, SGCB, LGMD2E,SGCD, SGD, LGMD2F, CMD1L, TCAP, LGMD2G, CMD1N, TRIM32, HT2A, LGMD2H,FKRP, MDC1C, LGMD2I, TTN, CMD1G, TMD, LGMD2J, POMT1, CAV3, LGMD1C,SEPN1, SELN, RSMD1, PLEC1, PLTN, EBS1); Osteopetrosis (LRP5, BMND1,LRP7, LR3, OPPG, VBCH2, CLCN7, CLC7, OPTA2, OSTM1, GL, TCIRG1, TIRC7,OC116, OPTB1); Muscular atrophy (VAPB, VAPC, ALS8, SMN1, SMA1, SMA2,SMA3, SMA4, BSCL2, SPG17, GARS, SMAD1, CMT2D, HEXB, IGHMBP2, SMUBP2,CATF1, SMARD1). Neurological and ALS (SOD1, ALS2, STEX, FUS, TARDBP,VEGF (VEGF-a, VEGF-b, neuronal diseases VEGF-c); Alzheimer disease (APP,AAA, CVAP, AD1, APOE, AD2, and disorders PSEN2, AD4, STM2, APBB2,FE65L1, NOS3, PLAU, URK, ACE, DCP1, ACE1, MPO, PACIP1, PAXIP1L, PTIP,A2M, BLMH, BMH, PSEN1, AD3); Autism (Mecp2, BZRAP1, MDGA2, Sema5A,Neurexin 1, GLO1, MECP2, RTT, PPMX, MRX16, MRX79, NLGN3, NLGN4,KIAA1260, AUTSX2); Fragile X Syndrome (FMR2, FXR1, FXR2, mGLUR5);Huntington's disease and disease like disorders (HD, IT15, PRNP, PRIP,JPH3, JP3, HDL2, TBP, SCA17); Parkinson disease (NR4A2, NURR1, NOT,TINUR, SNCAIP, TBP, SCA17, SNCA, NACP, PARK1, PARK4, DJ1, PARK7, LRRK2,PARK8, PINK1, PARK6, UCHL1, PARK5, SNCA, NACP, PARK1, PARK4, PRKN,PARK2, PDJ, DBH, NDUFV2); Rett syndrome (MECP2, RTT, PPMX, MRX16, MRX79,CDKL5, STK9, MECP2, RTT, PPMX, MRX16, MRX79, x-Synuclein, DJ-1);Schizophrenia (Neuregulin1 (Nrg1), Erb4 (receptor for Neuregulin),Complexin1 (Cplx1), Tph1 Tryptophan hydroxylase, Tph2, Tryptophanhydroxylase 2, Neurexin 1, GSK3, GSK3a, GSK3b, 5-HTT (Slc6a4), COMT, DRD(Drd1a), SLC6A3, DAOA, DTNBP1, Dao (Dao1)); Secretase Related Disorders(APH-1 (alpha and beta), Presenilin (Psen1), nicastrin, (Ncstn), PEN-2,Nos1, Parp1, Nat1, Nat2); Trinucleotide Repeat Disorders (HTT(Huntington's Dx), SBMA/SMAX1/AR (Kennedy's Dx), FXN/X25 (Friedrich'sAtaxia), ATX3 (Machado- Joseph's Dx), ATXN1 and ATXN2 (spinocerebellarataxias), DMPK (myotonic dystrophy), Atrophin-1 and Atn1 (DRPLA Dx), CBP(Creb-BP - global instability), VLDLR (Alzheimer's), Atxn7, Atxn10).Occular diseases and Age-related macular degeneration (Abcr, Ccl2, Cc2,cp (ceruloplasmin), disorders Timp3, cathepsinD, Vldlr, Ccr2); Cataract(CRYAA, CRYA1, CRYBB2, CRYB2, PITX3, BFSP2, CP49, CP47, CRYAA, CRYA1,PAX6, AN2, MGDA, CRYBA1, CRYB1, CRYGC, CRYG3, CCL, LIM2, MP19, CRYGD,CRYG4, BFSP2, CP49, CP47, HSF4, CTM, HSF4, CTM, MIP, AQP0, CRYAB, CRYA2,CTPP2, CRYBB1, CRYGD, CRYG4, CRYBB2, CRYB2, CRYGC, CRYG3, CCL, CRYAA,CRYA1, GJA8, CX50, CAE1, GJA3, CX46, CZP3, CAE3, CCM1, CAM, KRIT1);Corneal clouding and dystrophy (APOA1, TGFBI, CSD2, CDGG1, CSD, BIGH3,CDG2, TACSTD2, TROP2, M1S1, VSX1, RINX, PPCD, PPD, KTCN, COL8A2, FECD,PPCD2, PIP5K3, CFD); Cornea plana congenital (KERA, CNA2); Glaucoma(MYOC, TIGR, GLC1A, JOAG, GPOA, OPTN, GLC1E, FIP2, HYPL, NRP, CYP1B1,GLC3A, OPA1, NTG, NPG, CYP1B1, GLC3A); Leber congenital amaurosis (CRB1,RP12, CRX, CORD2, CRD, RPGRIP1, LCA6, CORD9, RPE65, RP20, AIPL1, LCA4,GUCY2D, GUC2D, LCA1, CORD6, RDH12, LCA3); Macular dystrophy (ELOVL4,ADMD, STGD2, STGD3, RDS, RP7, PRPH2, PRPH, AVMD, AOFMD, VMD2).

TABLE 12 CELLULAR FUNCTION GENES PI3K/AKT Signaling PRKCE; ITGAM; ITGA5;IRAK1; PRKAA2; EIF2AK2; PTEN; EIF4E; PRKCZ; GRK6; MAPK1; TSC1; PLK1;AKT2; IKBKB; PIK3CA; CDK8; CDKN1B; NFKB2; BCL2; PIK3CB; PPP2R1A; MAPK8;BCL2L1; MAPK3; TSC2; ITGA1; KRAS; EIF4EBP1; RELA; PRKCD; NOS3; PRKAA1;MAPK9; CDK2; PPP2CA; PIM1; ITGB7; YWHAZ; ILK; TP53; RAF1; IKBKG; RELB;DYRK1A; CDKN1A; ITGB1; MAP2K2; JAK1; AKT1; JAK2; PIK3R1; CHUK; PDPK1;PPP2R5C; CTNNB1; MAP2K1; NFKB1; PAK3; ITGB3; CCND1; GSK3A; FRAP1; SFN;ITGA2; TTK; CSNK1A1; BRAF; GSK3B; AKT3; FOXO1; SGK; HSP90AA1; RPS6KB1ERK/MAPK Signaling PRKCE; ITGAM; ITGA5; HSPB1; IRAK1; PRKAA2; EIF2AK2;RAC1; RAP1A; TLN1; EIF4E; ELK1; GRK6; MAPK1; RAC2; PLK1; AKT2; PIK3CA;CDK8; CREB1; PRKCI; PTK2; FOS; RPS6KA4; PIK3CB; PPP2R1A; PIK3C3; MAPK8;MAPK3; ITGA1; ETS1; KRAS; MYCN; EIF4EBP1; PPARG; PRKCD; PRKAA1; MAPK9;SRC; CDK2; PPP2CA; PIM1; PIK3C2A; ITGB7; YWHAZ; PPP1CC; KSR1; PXN; RAF1;FYN; DYRK1A; ITGB1; MAP2K2; PAK4; PIK3R1; STAT3; PPP2R5C; MAP2K1; PAK3;ITGB3; ESR1; ITGA2; MYC; TTK; CSNK1A1; CRKL; BRAF; ATF4; PRKCA; SRF;STAT1; SGK Glucocorticoid Receptor RAC1; TAF4B; EP300; SMAD2; TRAF6;PCAF; ELK1; Signaling MAPK1; SMAD3; AKT2; IKBKB; NCOR2; UBE2I; PIK3CA;CREB1; FOS; HSPA5; NFKB2; BCL2; MAP3K14; STAT5B; PIK3CB; PIK3C3; MAPK8;BCL2L1; MAPK3; TSC22D3; MAPK10; NRIP1; KRAS; MAPK13; RELA; STAT5A;MAPK9; NOS2A; PBX1; NR3C1; PIK3C2A; CDKN1C; TRAF2; SERPINE1; NCOA3;MAPK14; TNF; RAF1; IKBKG; MAP3K7; CREBBP; CDKN1A; MAP2K2; JAK1; IL8;NCOA2; AKT1; JAK2; PIK3R1; CHUK; STAT3; MAP2K1; NFKB1; TGFBR1; ESR1;SMAD4; CEBPB; JUN; AR; AKT3; CCL2; MMP1; STAT1; IL6; HSP90AA1 AxonalGuidance PRKCE; ITGAM; ROCK1; ITGA5; CXCR4; ADAM12; Signaling IGF1;RAC1; RAP1A; E1F4E; PRKCZ; NRP1; NTRK2; ARHGEF7; SMO; ROCK2; MAPK1; PGF;RAC2; PTPN11; GNAS; AKT2; PIK3CA; ERBB2; PRKCI; PTK2; CFL1; GNAQ;PIK3CB; CXCL12; PIK3C3; WNT11; PRKD1; GNB2L1; ABL1; MAPK3; ITGA1; KRAS;RHOA; PRKCD; PIK3C2A; ITGB7; GLI2; PXN; VASP; RAF1; FYN; ITGB1; MAP2K2;PAK4; ADAM17; AKT1; PIK3R1; GLI1; WNT5A; ADAM10; MAP2K1; PAK3; ITGB3;CDC42; VEGFA; ITGA2; EPHA8; CRKL; RND1; GSK3B; AKT3; PRKCA EphrinReceptor PRKCE; ITGAM; ROCK1; ITGA5; CXCR4; IRAK1; Signaling PRKAA2;EIF2AK2; RAC1; RAP1A; GRK6; ROCK2; MAPK1; PGF; RAC2; PTPN11; GNAS; PLK1;AKT2; DOK1; CDK8; CREB1; PTK2; CFL1; GNAQ; MAP3K14; CXCL12; MAPK8;GNB2L1; ABL1; MAPK3; ITGA1; KRAS; RHOA; PRKCD; PRKAA1; MAPK9; SRC; CDK2;PIM1; ITGB7; PXN; RAF1; FYN; DYRK1A; ITGB1; MAP2K2; PAK4, AKT1; JAK2;STAT3; ADAM10; MAP2K1; PAK3; ITGB3; CDC42; VEGFA; ITGA2; EPHA8; TTK;CSNK1A1; CRKL; BRAF; PTPN13; ATF4; AKT3; SGK Actin Cytoskeleton ACTN4;PRKCE; ITGAM; ROCK1; ITGA5; IRAK1; Signaling PRKAA2; EIF2AK2; RAC1; INS;ARHGEF7; GRK6; ROCK2; MAPK1; RAC2; PLK1; AKT2; PIK3CA; CDK8; PTK2; CFL1;PIK3CB; MYH9; DIAPH1; PIK3C3; MAPK8; F2R; MAPK3; SLC9A1; ITGA1; KRAS;RHOA; PRKCD; PRKAA1; MAPK9; CDK2; PIM1; PIK3C2A; ITGB7; PPP1CC; PXN;VIL2; RAF1; GSN; DYRK1A; ITGB1; MAP2K2; PAK4; PIP5K1A; PIK3R1; MAP2K1;PAK3; ITGB3; CDC42; APC; ITGA2; TTK; CSNK1A1; CRKL; BRAF; VAV3; SGKHuntington's Disease PRKCE; IGF1; EP300; RCOR1; PRKCZ; HDAC4; TGM2;Signaling MAPK1; CAPNS1; AKT2; EGFR; NCOR2; SP1; CAPN2; PIK3CA; HDAC5;CREB1; PRKC1; HSPA5; REST; GNAQ; PIK3CB; PIK3C3; MAPK8; IGF1R; PRKD1;GNB2L1; BCL2L1; CAPN1; MAPK3; CASP8; HDAC2; HDAC7A; PRKCD; HDAC11;MAPK9; HDAC9; PIK3C2A; HDAC3; TP53; CASP9; CREBBP; AKT1; PIK3R1; PDPK1;CASP1; APAF1; FRAP1; CASP2; JUN; BAX; ATF4; AKT3; PRKCA; CLTC; SGK;HDAC6; CASP3 Apoptosis Signaling PRKCE; ROCK1; BID; IRAK1; PRKAA2;EIF2AK2; BAK1; BIRC4; GRK6; MAPK1; CAPNS1; PLK1; AKT2; IKBKB; CAPN2;CDK8; FAS; NFKB2; BCL2; MAP3K14; MAPK8; BCL2L1; CAPN1; MAPK3; CASP8;KRAS; RELA; PRKCD; PRKAA1; MAPK9; CDK2; PIM1; TP53; TNF; RAF1; IKBKG;RELB; CASP9; DYRK1A; MAP2K2; CHUK; APAF1; MAP2K1; NFKB1; PAK3; LMNA;CASP2; BIRC2; TTK; CSNK1A1; BRAF; BAX; PRKCA; SGK; CASP3; BIRC3; PARP1 BCell Receptor RAC1; PTEN; LYN; ELK1; MAPK1; RAC2; PTPN11; SignalingAKT2; IKBKB; PIK3CA; CREB1; SYK; NFKB2; CAMK2A; MAP3K14; PIK3CB; PIK3C3;MAPK8; BCL2L1; ABL1; MAPK3; ETS1; KRAS; MAPK13; RELA; PTPN6; MAPK9;EGR1; PIK3C2A; BTK; MAPK14; RAF1; IKBKG; RELB; MAP3K7; MAP2K2; AKT1;PIK3R1; CHUK; MAP2K1; NFKB1; CDC42; GSK3A; FRAP1; BCL6; BCL10; JUN;GSK3B; ATF4; AKT3; VAV3; RPS6KB1 Leukocyte Extravasation ACTN4; CD44;PRKCE; ITGAM; ROCK1; CXCR4; CYBA; Signaling RAC1; RAP1A; PRKCZ; ROCK2;RAC2; PTPN11; MMP14; PIK3CA; PRKCI; PTK2; PIK3CB; CXCL12; PIK3C3; MAPK8;PRKD1; ABL1; MAPK10; CYBB; MAPK13; RHOA; PRKCD; MAPK9; SRC; PIK3C2A;BTK; MAPK14; NOX1; PXN; VIL2; VASP; ITGB1; MAP2K2; CTNND1; PIK3R1;CTNNB1; CLDN1; CDC42; F11R; ITK; CRKL; VAV3; CTTN; PRKCA; MMP1; MMP9Integrin Signaling ACTN4; ITGAM; ROCK1; ITGA5; RAC1; PTEN; RAP1A; TLN1;ARHGEF7; MAPK1; RAC2; CAPNS1; AKT2; CAPN2; PIK3CA; PTK2; PIK3CB; PIK3C3;MAPK8; CAV1; CAPN1; ABL1; MAPK3; ITGA1; KRAS; RHOA; SRC; PIK3C2A; ITGB7;PPP1CC; ILK; PXN; VASP; RAF1; FYN; ITGB1; MAP2K2; PAK4; AKT1; PIK3R1;TNK2; MAP2K1; PAK3; ITGB3; CDC42; RND3; ITGA2; CRKL; BRAF; GSK3B; AKT3Acute Phase Response IRAK1; SOD2; MYD88; TRAF6; ELK1; MAPK1; PTPN11;Signaling AKT2; IKBKB; PIK3CA; FOS; NFKB2; MAP3K14; PIK3CB; MAPK8;RIPK1; MAPK3; IL6ST; KRAS; MAPK13; IL6R; RELA; SOCS1; MAPK9; FTL; NR3C1;TRAF2; SERPINE1; MAPK14; TNF; RAF1; PDK1; IKBKG; RELB; MAP3K7; MAP2K2;AKT1; JAK2; PIK3R1; CHUK; STAT3; MAP2K1; NFKB1; FRAP1; CEBPB; JUN; AKT3;IL1R1; IL6 PTEN Signaling ITGAM; ITGA5; RAC1; PTEN; PRKCZ; BCL2L11;MAPK1; RAC2; AKT2; EGFR; IKBKB; CBL; PIK3CA; CDKN1B; PTK2; NFKB2; BCL2;PIK3CB; BCL2L1; MAPK3; ITGA1; KRAS; ITGB7; ILK; PDGFRB; INSR; RAF1;IKBKG; CASP9; CDKN1A; ITGB1; MAP2K2; AKT1; PIK3R1; CHUK; PDGFRA; PDPK1;MAP2K1; NFKB1; ITGB3; CDC42; CCND1; GSK3A; ITGA2; GSK3B; AKT3; FOXO1;CASP3; RPS6KB1 p53 Signaling PTEN; EP300; BBC3; PCAF; FASN; BRCA1;GADD45A; BIRC5; AKT2; PIK3CA; CHEK1; TP53INP1; BCL2; PIK3CB; PIK3C3;MAPK8; THBS1; ATR; BCL2L1; E2F1; PMAIP1; CHEK2; TNFRSF10B; TP73; RB1;HDAC9; CDK2; PIK3C2A; MAPK14; TP53; LRDD; CDKN1A; HIPK2; AKT1; PIK3R1;RRM2B; APAF1; CTNNB1; SIRT1; CCND1; PRKDC; ATM; SFN; CDKN2A; JUN; SNAI2;GSK3B; BAX; AKT3 Aryl Hydrocarbon HSPB1; EP300; FASN; TGM2; RXRA; MAPK1;NQO1; Receptor NCOR2; SP1; ARNT; CDKN1B; FOS; CHEK1; Signaling SMARCA4;NFKB2; MAPK8; ALDH1A1; ATR; E2F1; MAPK3; NRIP1; CHEK2; RELA; TP73;GSTP1; RB1; SRC; CDK2; AHR; NFE2L2; NCOA3; TP53; TNF; CDKN1A; NCOA2;APAF1; NFKB1; CCND1; ATM; ESR1; CDKN2A; MYC; JUN; ESR2; BAX; IL6;CYP1B1; HSP90AA1 Xenobiotic Metabolism PRKCE; EP300; PRKCZ; RXRA; MAPK1;NQO1; Signaling NCOR2; PIK3CA; ARNT; PRKCI; NFKB2; CAMK2A; PIK3CB;PPP2R1A; PIK3C3; MAPK8; PRKD1; ALDH1A1; MAPK3; NRIP1; KRAS; MAPK13;PRKCD; GSTP1; MAPK9; NOS2A; ABCB1; AHR; PPP2CA; FTL; NFE2L2; PIK3C2A;PPARGC1A; MAPK14; TNF; RAF1; CREBBP; MAP2K2; PIK3R1; PPP2R5C; MAP2K1;NFKB1; KEAP1; PRKCA; EIF2AK3; IL6; CYP1B1; HSP90AA1 SAPK/JNK SignalingPRKCE; IRAK1; PRKAA2; EIF2AK2; RAC1; ELK1; GRK6; MAPK1; GADD45A; RAC2;PLK1; AKT2; PIK3CA; FADD; CDK8; PIK3CB; PIK3C3; MAPK8; RIPK1; GNB2L1;IRS1; MAPK3; MAPK10; DAXX; KRAS; PRKCD; PRKAA1; MAPK9; CDK2; PIM1;PIK3C2A; TRAF2; TP53; LCK; MAP3K7; DYRK1A; MAP2K2; PIK3R1; MAP2K1; PAK3;CDC42; JUN; TTK; CSNK1A1; CRKL; BRAF; SGK PPAr/RXR Signaling PRKAA2;EP300; INS; SMAD2; TRAF6; PPARA; FASN; RXRA; MAPK1; SMAD3; GNAS; IKBKB;NCOR2; ABCA1; GNAQ; NFKB2; MAP3K14; STAT5B; MAPK8; IRS1; MAPK3; KRAS;RELA; PRKAA1; PPARGC1A; NCOA3; MAPK14; INSR; RAF1; IKBKG; RELB; MAP3K7;CREBBP; MAP2K2; JAK2; CHUK; MAP2K1; NFKB1; TGFBR1; SMAD4; JUN; IL1R1;PRKCA; IL6; HSP90AA1; ADIPOQ NF-KB Signaling IRAK1; EIF2AK2; EP300; INS;MYD88; PRKCZ: TRAF6; TBK1; AKT2; EGFR; IKBKB; PIK3CA; BTRC; NFKB2;MAP3K14; PIK3CB; PIK3C3; MAPK8; RIPK1; HDAC2; KRAS; RELA; PIK3C2A;TRAF2; TLR4: PDGFRB; TNF; INSR; LCK; IKBKG; RELB; MAP3K7; CREBBP; AKT1;PIK3R1; CHUK; PDGFRA; NFKB1; TLR2; BCL10; GSK3B; AKT3; TNFAIP3; IL1R1Neuregulin Signaling ERBB4; PRKCE; ITGAM; ITGA5: PTEN; PRKCZ; ELK1;MAPK1; PTPN11; AKT2; EGFR; ERBB2; PRKCI; CDKN1B; STAT5B; PRKD1; MAPK3;ITGA1; KRAS; PRKCD; STAT5A; SRC; ITGB7; RAF1; ITGB1; MAP2K2; ADAM17;AKT1; PIK3R1; PDPK1; MAP2K1; ITGB3; EREG; FRAP1; PSEN1; ITGA2; MYC;NRG1; CRKL; AKT3; PRKCA; HSP90AA1; RPS6KB1 Wnt & Beta catenin CD44;EP300; LRP6; DVL3; CSNK1E; GJA1; SMO; Signaling AKT2; PIN1; CDH1; BTRC;GNAQ; MARK2; PPP2R1A; WNT11; SRC; DKK1; PPP2CA; SOX6; SFRP2: ILK; LEF1;SOX9; TP53; MAP3K7; CREBBP; TCF7L2; AKT1; PPP2R5C; WNT5A; LRP5; CTNNB1;TGFBR1; CCND1; GSK3A; DVL1; APC; CDKN2A; MYC; CSNK1A1; GSK3B; AKT3; SOX2Insulin Receptor PTEN; INS; EIF4E; PTPN1; PRKCZ; MAPK1; TSC1; SignalingPTPN11; AKT2; CBL; PIK3CA; PRKCI; PIK3CB; PIK3C3; MAPK8; IRS1; MAPK3;TSC2; KRAS; EIF4EBP1; SLC2A4; PIK3C2A; PPP1CC; INSR; RAF1; FYN; MAP2K2;JAK1; AKT1; JAK2; PIK3R1; PDPK1; MAP2K1; GSK3A; FRAP1; CRKL; GSK3B;AKT3; FOXO1; SGK; RPS6KB1 IL-6 Signaling HSPB1; TRAF6; MAPKAPK2; ELK1;MAPK1; PTPN11; IKBKB; FOS; NFKB2: MAP3K14; MAPK8; MAPK3; MAPK10; IL6ST;KRAS; MAPK13; IL6R; RELA; SOCS1; MAPK9; ABCB1; TRAF2; MAPK14; TNF; RAF1;IKBKG; RELB; MAP3K7; MAP2K2; IL8; JAK2; CHUK; STAT3; MAP2K1; NFKB1;CEBPB; JUN; IL1R1; SRF; IL6 Hepatic Cholestasis PRKCE; IRAK1; INS;MYD88; PRKCZ; TRAF6; PPARA; RXRA; IKBKB; PRKCI; NFKB2; MAP3K14; MAPK8;PRKD1; MAPK10; RELA; PRKCD; MAPK9; ABCB1; TRAF2; TLR4; TNF; INSR; IKBKG;RELB; MAP3K7; IL8; CHUK; NR1H2; TJP2; NFKB1; ESR1; SREBF1; FGFR4; JUN;IL1R1; PRKCA; IL6 IGF-1 Signaling IGF1; PRKCZ; ELK1; MAPK1; PTPN11;NEDD4; AKT2; PIK3CA; PRKCI; PTK2; FOS; PIK3CB; PIK3C3; MAPK8; IGF1R;IRS1; MAPK3; IGFBP7; KRAS; PIK3C2A; YWHAZ; PXN; RAF1; CASP9; MAP2K2;AKT1; PIK3R1; PDPK1; MAP2K1; IGFBP2; SFN; JUN; CYR61; AKT3; FOXO1; SRF;CTGF; RPS6KB1 NRF2-mediated PRKCE; EP300; SOD2; PRKCZ; MAPK1; SQSTM1;Oxidative NQO1; PIK3CA; PRKCI; FOS; PIK3CB; PIK3C3; MAPK8; StressResponse PRKD1; MAPK3; KRAS; PRKCD; GSTP1; MAPK9; FTL; NFE2L2; PIK3C2A;MAPK14; RAF1; MAP3K7; CREBBP; MAP2K2; AKT1; PIK3R1; MAP2K1; PPIB; JUN;KEAP1; GSK3B; ATF4; PRKCA; EIF2AK3; HSP90AA1 Hepatic Fibrosis/HepaticEDN1; IGF1; KDR; FLT1; SMAD2; FGFR1; MET; PGF; Stellate Cell ActivationSMAD3; EGFR; FAS; CSF1; NFKB2; BCL2; MYH9; IGF1R; IL6R; RELA; TLR4;PDGFRB; TNF; RELB; IL8; PDGFRA; NFKB1; TGFBR1; SMAD4; VEGFA; BAX; IL1R1;CCL2; HGF; MMP1; STAT1; IL6; CTGF; MMP9 PPAR Signaling EP300; INS;TRAF6; PPARA; RXRA; MAPK1; IKBKB; NCOR2; FOS; NFKB2; MAP3K14; STAT5B;MAPK3; NRIP1; KRAS; PPARG; RELA; STAT5A; TRAF2; PPARGC1A; PDGFRB; TNF;INSR; RAF1; IKBKG; RELB; MAP3K7; CREBBP; MAP2K2; CHUK; PDGFRA; MAP2K1;NFKB1; JUN; IL1R1; HSP90AA1 Fc Epsilon RI Signaling PRKCE; RAC1; PRKCZ;LYN; MAPK1; RAC2; PTPN11; AKT2; PIK3CA; SYK; PRKCI; PIK3CB; PIK3C3;MAPK8; PRKD1; MAPK3; MAPK10; KRAS; MAPK13; PRKCD; MAPK9; PIK3C2A; BTK;MAPK14; TNF; RAF1; FYN; MAP2K2; AKT1; PIK3R1; PDPK1; MAP2K1; AKT3; VAV3;PRKCA G-Protein Coupled PRKCE; RAP1A; RGS16; MAPK1; GNAS; AKT2; IKBKB;Receptor Signaling PIK3CA; CREB1; GNAQ; NFKB2; CAMK2A; PIK3CB; PIK3C3;MAPK3; KRAS; RELA; SRC; PIK3C2A; RAF1; IKBKG; RELB; FYN; MAP2K2; AKT1;PIK3R1; CHUK; PDPK1; STAT3; MAP2K1; NFKB1; BRAF; ATF4; AKT3; PRKCAInositol Phosphate PRKCE; IRAK1; PRKAA2; EIF2AK2; PTEN; GRK6; MetabolismMAPK1; PLK1; AKT2; PIK3CA; CDK8; PIK3CB; PIK3C3; MAPK8; MAPK3; PRKCD;PRKAA1; MAPK9; CDK2; PIM1; PIK3C2A; DYRK1A; MAP2K2; PIP5K1A; PIK3R1;MAP2K1; PAK3; ATM; TTK; CSNK1A1; BRAF; SGK PDGF Signaling EIF2AK2; ELK1;ABL2; MAPK1; PIK3CA; FOS; PIK3CB; PIK3C3; MAPK8; CAV1; ABL1; MAPK3;KRAS; SRC; PIK3C2A; PDGFRB; RAF1; MAP2K2; JAK1; JAK2; PIK3R1; PDGFRA;STAT3; SPHK1; MAP2K1; MYC; JUN; CRKL; PRKCA; SRF; STAT1; SPHK2 VEGFSignaling ACTN4; ROCK1; KDR; FLT1; ROCK2; MAPK1; PGF; AKT2; PIK3CA;ARNT; PTK2; BCL2; PIK3CB; PIK3C3; BCL2L1; MAPK3; KRAS; HIF1A; NOS3;PIK3C2A; PXN; RAF1; MAP2K2; ELAVL1; AKT1; PIK3R1; MAP2K1; SFN; VEGFA;AKT3; FOXO1; PRKCA Natural Killer Cell PRKCE; RAC1; PRKCZ; MAPK1; RAC2;PTPN11; Signaling KIR2DL3; AKT2; PIK3CA; SYK; PRKCI; PIK3CB; PIK3C3;PRKD1; MAPK3; KRAS; PRKCD; PTPN6; PIK3C2A; LCK; RAF1; FYN; MAP2K2; PAK4;AKT1; PIK3R1; MAP2K1; PAK3; AKT3; VAV3; PRKCA Cell Cycle: G1/S HDAC4;SMAD3; SUV39H1; HDAC5; CDKN1B; BTRC; Checkpoint Regulation ATR; ABL1;E2F1; HDAC2; HDAC7A; RB1; HDAC11; HDAC9; CDK2; E2F2; HDAC3; TP53;CDKN1A; CCND1; E2F4; ATM; RBL2; SMAD4; CDKN2A; MYC; NRG1; GSK3B; RBL1;HDAC6 T Cell Receptor RAC1; ELK1; MAPK1; IKBKB; CBL; PIK3CA; FOS;Signaling NFKB2; PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS; RELA, PIK3C2A; BTK;LCK; RAF1; IKBKG; RELB, FYN; MAP2K2; PIK3R1; CHUK; MAP2K1; NFKB1; ITK;BCL10; JUN; VAV3 Death Receptor Signaling CRADD; HSPB1; BID; BIRC4;TBK1; IKBKB; FADD; FAS; NFKB2; BCL2; MAP3K14; MAPK8; RIPK1; CASP8; DAXX;TNFRSF10B; RELA; TRAF2; TNF; IKBKG; RELB; CASP9; CHUK; APAF1; NFKB1;CASP2; BIRC2; CASP3; BIRC3 FGF Signaling RAC1; FGFR1; MET; MAPKAPK2;MAPK1; PTPN11; AKT2; PIK3CA; CREB1; PIK3CB; PIK3C3; MAPK8; MAPK3;MAPK13; PTPN6; PIK3C2A; MAPK14; RAF1; AKT1; PIK3R1; STAT3; MAP2K1;FGFR4; CRKL; ATF4; AKT3; PRKCA; HGF GM-CSF Signaling LYN; ELK1; MAPK1;PTPN11; AKT2; PIK3CA; CAMK2A; STAT5B; PIK3CB; PIK3C3; GNB2L1; BCL2L1;MAPK3; ETS1; KRAS; RUNX1; PIM1; PIK3C2A; RAF1; MAP2K2; AKT1; JAK2;PIK3R1; STAT3; MAP2K1; CCND1; AKT3; STAT1 Amyotrophic Lateral BID; IGF1;RAC1; BIRC4; PGF; CAPNS1; CAPN2; Sclerosis Signaling PIK3CA; BCL2;PIK3CB; PIK3C3; BCL2L1; CAPN1; PIK3C2A; TP53; CASP9; PIK3R1; RAB5A;CASP1; APAF1; VEGFA; BIRC2; BAX; AKT3; CASP3; BIRC3 JAK/Stat SignalingPTPN1; MAPK1; PTPN11; AKT2; PIK3CA; STAT5B; PIK3CB; PIK3C3; MAPK3; KRAS;SOCS1; STAT5A; PTPN6; PIK3C2A; RAF1; CDKN1A; MAP2K2; JAK1; AKT1; JAK2;PIK3R1; STAT3; MAP2K1; FRAP1; AKT3; STAT1 Nicotinate and PRKCE; IRAK1;PRKAA2; EIF2AK2; GRK6; MAPK1; Nicotinamide PLK1; AKT2; CDK8; MAPK8;MAPK3; PRKCD; PRKAA1; Metabolism PBEF1; MAPK9; CDK2; PIM1; DYRK1A;MAP2K2; MAP2K1; PAK3; NT5E; TTK; CSNK1A1; BRAF; SGK Chemokine SignalingCXCR4; ROCK2; MAPK1; PTK2; FOS; CFL1; GNAQ; CAMK2A; CXCL12; MAPK8;MAPK3; KRAS; MAPK13; RHOA; CCR3; SRC; PPP1CC; MAPK14; NOX1; RAF1;MAP2K2; MAP2K1; JUN; CCL2; PRKCA IL-2 Signaling ELK1; MAPK1; PTPN11;AKT2; PIK3CA; SYK; FOS; STAT5B; PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS;SOCS1; STAT5A; PIK3C2A; LCK; RAF1; MAP2K2; JAK1; AKT1; PIK3R1; MAP2K1;JUN; AKT3 Synaptic Long Term PRKCE; IGF1; PRKCZ; PRDX6; LYN; MAPK1;GNAS; Depression PRKCI; GNAQ; PPP2R1A; IGF1R; PRKD1; MAPK3; KRAS; GRN;PRKCD; NOS3; NOS2A; PPP2CA; YWHAZ; RAF1; MAP2K2; PPP2R5C; MAP2K1; PRKCAEstrogen Receptor TAF4B; EP300; CARM1; PCAF; MAPK1; NCOR2; SignalingSMARCA4; MAPK3; NRIP1; KRAS; SRC; NR3C1; HDAC3; PPARGC1A; RBM9; NCOA3;RAF1; CREBBP; MAP2K2; NCOA2; MAP2K1; PRKDC; ESR1; ESR2 ProteinUbiquitination TRAF6; SMURF1; BIRC4; BRCA1; UCHL1; NEDD4; Pathway CBL;UBE2I; BTRC; HSPA5; USP7; USP10; FBXW7; USP9X; STUB1; USP22; B2M; BIRC2;PARK2; USP8; USP1; VHL; HSP90AA1; BIRC3 IL-10 Signaling TRAF6; CCR1;ELK1; IKBKB; SP1; FOS; NFKB2; MAP3K14; MAPK8; MAPK13; RELA; MAPK14; TNF;IKBKG; RELB; MAP3K7; JAK1; CHUK; STAT3; NFKB1; JUN; IL1R1; IL6 VDR/RXRActivation PRKCE; EP300; PRKCZ; RXRA; GADD45A; HES1; NCOR2; SP1; PRKC1;CDKN1B; PRKD1; PRKCD; RUNX2; KLF4; YY1; NCOA3; CDKN1A; NCOA2; SPP1;LRP5; CEBPB; FOXO1; PRKCA TGF-beta Signaling EP300; SMAD2; SMURF1;MAPK1; SMAD3; SMAD1; FOS; MAPK8; MAPK3; KRAS; MAPK9; RUNX2; SERPINE1;RAF1; MAP3K7; CREBBP; MAP2K2; MAP2K1; TGFBR1; SMAD4; JUN; SMAD5Toll-like Receptor IRAK1; EIF2AK2; MYD88; TRAF6; PPARA; ELK1; SignalingIKBKB; FOS; NFKB2; MAP3K14; MAPK8; MAPK13; RELA; TLR4; MAPK14; IKBKG;RELB; MAP3K7; CHUK; NFKB1; TLR2; JUN p38 MAPK Signaling HSPB1; IRAK1;TRAF6; MAPKAPK2; ELK1; FADD; FAS; CREB1; DDIT3; RPS6KA4; DAXX; MAPK13;TRAF2; MAPK14; TNF; MAP3K7; TGFBR1; MYC; ATF4; IL1R1; SRF; STAT1Neurotrophin/TRK NTRK2; MAPK1; PTPN11; PIK3CA; CREB1; FOS; SignalingPIK3CB; PIK3C3; MAPK8; MAPK3; KRAS; PIK3C2A; RAF1; MAP2K2; AKT1; PIK3R1;PDPK1; MAP2K1; CDC42; JUN; ATF4 FXR/RXR Activation INS; PPARA; FASN;RXRA; AKT2; SDC1; MAPK8; APOB; MAPK10; PPARG; MTTP; MAPK9; PPARGC1A;TNF; CREBBP; AKT1; SREBF1; FGFR4; AKT3; FOXO1 Synaptic Long Term PRKCE;RAP1A; EP300; PRKCZ; MAPK1; CREB1; Potentiation PRKCI; GNAQ; CAMK2A;PRKD1; MAPK3; KRAS; PRKCD; PPP1CC; RAF1; CREBBP; MAP2K2; MAP2K1; ATF4;PRKCA Calcium Signaling RAP1A; EP300; HDAC4; MAPK1; HDAC5; CREB1;CAMK2A; MYH9; MAPK3; HDAC2; HDAC7A; HDAC11; HDAC9; HDAC3; CREBBP; CALR;CAMKK2; ATF4; HDAC6 EGF Signaling ELK1; MAPK1; EGFR; PIK3CA; FOS;PIK3CB; PIK3C3; MAPK8; MAPK3; PIK3C2A; RAF1; JAK1; PIK3R1; STAT3;MAP2K1; JUN; PRKCA; SRF; STAT1 Hypoxia Signaling in the EDN1; PTEN;EP300; NQO1; UBE2I; CREB1; ARNT; Cardiovascular System HIF1A; SLC2A4;NOS3; TP53; LDHA; AKT1; ATM; VEGFA; JUN; ATF4; VHL; HSP90AA1 LPS/IL-1Mediated IRAK1; MYD88; TRAF6; PPARA; RXRA; ABCA1, Inhibition MAPK8;ALDH1A1; GSTP1; MAPK9; ABCB1; TRAF2; of RXR Function TLR4; TNF; MAP3K7;NR1H2; SREBF1; JUN; IL1R1 LXR/RXR Activation FASN; RXRA; NCOR2; ABCA1;NFKB2; IRF3; RELA; NOS2A; TLR4; TNF; RELB; LDLR; NR1H2; NFKB1; SREBF1;IL1R1; CCL2; IL6; MMP9 Amyloid Processing PRKCE; CSNK1E; MAPK1; CAPNS1;AKT2; CAPN2; CAPN1; MAPK3; MAPK13; MAPT; MAPK14; AKT1; PSEN1; CSNK1A1;GSK3B; AKT3; APP IL-4 Signaling AKT2; PIK3CA; PIK3CB; PIK3C3; IRS1;KRAS; SOCS1; PTPN6; NR3C1; PIK3C2A; JAK1; AKT1; JAK2; PIK3R1; FRAP1;AKT3; RPS6KB1 Cell Cycle: G2/M DNA EP300; PCAF; BRCA1; GADD45A; PLK1;BTRC; Damage Checkpoint CHEK1; ATR; CHEK2; YWHAZ; TP53; CDKN1A;Regulation PRKDC; ATM; SFN; CDKN2A Nitric Oxide Signaling in KDR; FLT1;PGF; AKT2; PIK3CA; PIK3CB; PIK3C3; the Cardiovascular System CAV1;PRKCD; NOS3; PIK3C2A; AKT1; PIK3R1; VEGFA; AKT3; HSP90AA1 PurineMetabolism NME2; SMARCA4; MYH9; RRM2; ADAR; EIF2AK4; PKM2; ENTPD1;RAD51; RRM2B; TJP2; RAD51C; NT5E; POLD1; NME1 cAMP-mediated RAP1A;MAPK1; GNAS; CREB1; CAMK2A; MAPK3; Signaling SRC; RAF1; MAP2K2; STAT3;MAP2K1; BRAF; ATF4 Mitochondrial SOD2; MAPK8; CASP8; MAPK10; MAPK9;CASP9; Dysfunction PARK7; PSEN1; PARK2; APP; CASP3 Notch Signaling HES1;JAG1; NUMB; NOTCH4; ADAM17; NOTCH2; PSEN1; NOTCH3; NOTCH1; DLL4Endoplasmic Reticulum HSPA5; MAPK8; XBP1; TRAF2; ATF6; CASP9; ATF4;Stress Pathway EIF2AK3; CASP3 Pyrimidine Metabolism NME2; AICDA; RRM2;EIF2AK4; ENTPD1; RRM2B; NT5E; POLD1; NME1 Parkinson's Signaling UCHL1;MAPK8; MAPK13; MAPK14; CASP9; PARK7; PARK2; CASP3 Cardiac & Beta GNAS;GNAQ; PPP2R1A; GNB2L1; PPP2CA; PPP1CC; Adrenergic Signaling PPP2R5CGlycolysis/ HK2; GCK; GPI; ALDH1A1; PKM2; LDHA; HK1 GluconeogenesisInterferon Signaling IRF1; SOCS1; JAK1; JAK2; IFITM1; STAT1; IFIT3 SonicHedgehog ARRB2; SMO; GLI2; DYRK1A; GLI1; GSK3B; DYRK1B SignalingGlycerophospholipid PLD1; GRN; GPAM; YWHAZ; SPHK1; SPHK2 MetabolismPhospholipid PRDX6; PLD1; GRN; YWHAZ; SPHK1; SPHK2 DegradationTryptophan Metabolism SIAH2; PRMT5; NEDD4; ALDH1A1; CYP1B1; SIAH1 LysineDegradation SUV39H1; EHMT2; NSD1; SETD7; PPP2R5C Nucleotide ExcisionERCC5; ERCC4; XPA; XPC; ERCC1 Repair Pathway Starch and Sucrose UCHL1;HK2; GCK; GPI; HK1 Metabolism Aminosugars Metabolism NQO1; HK2; GCK; HK1Arachidonic Acid PRDX6; GRN; YWHAZ; CYP1B1 Metabolism Circadian RhythmCSNK1E; CREB1; ATF4; NR1D1 Signaling Coagulation System BDKRB1; F2R;SERPINE1; F3 Dopamine Receptor PPP2R1A; PPP2CA; PPP1CC; PPP2R5CSignaling Glutathione Metabolism IDH2; GSTP1; ANPEP; IDH1 GlycerolipidMetabolism ALDH1A1; GPAM; SPHK1; SPHK2 Linoleic Acid Metabolism PRDX6;GRN; YWHAZ; CYP1B1 Methionine Metabolism DNMT1; DNMT3B; AHCY; DNMT3APyruvate Metabolism GLO1; ALDH1A1; PKM2; LDHA Arginine and ProlineALDH1A1; NOS3; NOS2A Metabolism Eicosanoid Signaling PRDX6; GRN; YWHAZFructose and Mannose HK2; GCK; HK1 Metabolism Galactose Metabolism HK2;GCK; HK1 Stilbene, Coumarine and PRDX6; PRDX1; TYR Lignin BiosynthesisAntigen Presentation CALR; B2M Pathway Biosynthesis of Steroids NQO1;DHCR7 Butanoate Metabolism ALDH1A1; NLGN1 Citrate Cycle IDH2; IDH1 FattyAcid Metabolism ALDH1A1; CYP1B1 Glycerophospholipid PRDX6; CHKAMetabolism Histidine Metabolism PRMT5; ALDH1A1 Inositol MetabolismERO1L; APEX1 Metabolism of GSTP1; CYP1B1 Xenobiotics by Cytochrome p450Methane Metabolism PRDX6; PRDX1 Phenylalanine PRDX6; PRDX1 MetabolismPropanoate Metabolism ALDH1A1; LDHA Selenoamino Acid PRMT5; AHCYMetabolism Sphingolipid Metabolism SPHK1; SPHK2 Aminophosphonate PRMT5Metabolism Androgen and Estrogen PRMT5 Metabolism Ascorbate and AldarateALDH1A1 Metabolism Bile Acid Biosynthesis ALDH1A1 Cysteine MetabolismLDHA Fatty Acid Biosynthesis FASN Glutamate Receptor GNB2L1 SignalingNRF2-mediated PRDX1 Oxidative Stress Response Pentose Phosphate GPIPathway Pentose and Glucuronate UCHL1 Interconversions RetinolMetabolism ALDH1A1 Riboflavin Metabolism TYR Tyrosine Metabolism PRMT5,TYR Ubiquinone Biosynthesis PRMT5 Valine, Leucine and ALDH1A1 IsoleucineDegradation Glycine, Serine and CHKA Threonine Metabolism LysineDegradation ALDH1A1 Pain/Taste TRPM5; TRPA1 Pain TRPM7; TRPC5; TRPC6;TRPC1; Cnr1; cnr2; Grk2; Trpa1; Pomc; Cgrp; Crf; Pka; Era; Nr2b; TRPM5;Prkaca; Prkacb; Prkar1a; Prkar2a Mitochondrial Function AIF; CytC; SMAC(Diablo); Aifm-1; Aifm-2 Developmental BMP-4; Chordin (Chrd); Noggin(Nog); WNT (Wnt2; Neurology Wnt2b; Wnt3a; Wnt4; Wnt5a; Wnt6; Wnt7b;Wnt8b; Wnt9a; Wnt9b; Wnt10a; Wnt10b; Wnt16); beta-catenin; Dkk-1;Frizzled related proteins; Otx-2; Gbx2; FGF-8; Reelin; Dab1; unc-86(Pou4f1 or Brn3a); Numb; Reln

Embodiments of the invention also relate to methods and compositionsrelated to knocking out genes, amplifying genes and repairing particularmutations associated with DNA repeat instability and neurologicaldisorders (Robert D. Wells, Tetsuo Ashizawa, Genetic Instabilities andNeurological Diseases, Second Edition, Academic Press, Oct. 13,2011-Medical). Specific aspects of tandem repeat sequences have beenfound to be responsible for more than twenty human diseases (Newinsights into repeat instability: role of RNA-DNA hybrids. McIvor E L,Polak U, Napierala M. RNA Biol. 2010 September-October; 7(5):551-8). Thepresent effector protein systems may be harnessed to correct thesedefects of genomic instability.

Several further aspects of the invention relate to correcting defectsassociated with a wide range of genetic diseases which are furtherdescribed on the website of the National Institutes of Health under thetopic subsection Genetic Disorders (website athealth.nih.gov/topic/GeneticDisorders). The genetic brain diseases mayinclude but are not limited to Adrenoleukodystrophy, Agenesis of theCorpus Callosum, Aicardi Syndrome, Alpers' Disease, Alzheimer's Disease,Barth Syndrome, Batten Disease, CADASIL, Cerebellar Degeneration,Fabry's Disease, Gerstmann-Straussler-Scheinker Disease, Huntington'sDisease and other Triplet Repeat Disorders, Leigh's Disease, Lesch-NyhanSyndrome, Menkes Disease, Mitochondrial Myopathies and NINDSColpocephaly. These diseases are further described on the website of theNational Institutes of Health under the subsection Genetic BrainDisorders.

Cas9 Development and Use

The present invention may be further illustrated and extended based onaspect of CRISPR-Cas9 development and use as set forth in the followingarticles hereby incorporated herein by reference and particularly asrelates to delivery of a CRISPR protein complex and uses of an RNAguided endonuclease in cells and organisms:

-   -   Multiplex genome engineering using CRISPR/Cas systems. Cong, L.,        Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.        D., Wu, X., Jiang, W., Marraffini, L. A., & Zhang, F. Science        February 15; 339(6121):819-23 (2013);    -   RNA-guided editing of bacterial genomes using CRISPR-Cas        systems. Jiang W., Bikard D., Cox D., Zhang F, Marraffini L A.        Nat Biotechnol March; 31(3):233-9 (2013);    -   One-Step Generation of Mice Carrying Mutations in Multiple Genes        by CRISPR/Cas-Mediated Genome Engineering. Wang H., Yang H.,        Shivalila C S., Dawlaty M M., Cheng A W., Zhang F., Jaenisch R.        Cell May 9; 153(4):910-8 (2013);    -   Optical control of mammalian endogenous transcription and        epigenetic states. Konermann S, Brigham M D, Trevino A E, Hsu P        D, Heidenreich M, Cong L, Platt R J, Scott D A, Church G M,        Zhang F. Nature. August 22; 500(7463):472-6. doi:        10.1038/Nature12466. Epub 2013 Aug. 23 (2013);    -   Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome        Editing Specificity. Ran, F A., Hsu, P D., Lin, C Y.,        Gootenberg, J S., Konermann, S., Trevino, A E., Scott, D A.,        Inoue, A., Matoba, S., Zhang, Y., & Zhang, F. Cell August 28.        pii: S0092-8674(13)01015-5 (2013-A);    -   DNA targeting specificity of RNA-guided Cas9 nucleases. Hsu, P.,        Scott, D., Weinstein, J., Ran, F A., Konermann, S., Agarwala,        V., Li, Y., Fine, E., Wu, X., Shalem, O., Cradick, T J.,        Marraffini, L A., Bao, G., & Zhang, F. Nat Biotechnol        doi:10.1038/nbt.2647 (2013);    -   Genome engineering using the CRISPR-Cas9 system. Ran, F A., Hsu,        P D., Wright, J., Agarwala, V., Scott, D A., Zhang, F. Nature        Protocols November; 8(11):2281-308 (2013-B);    -   Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells.        Shalem, O., Sanjana, N E., Hartenian, E., Shi, X., Scott, D A.,        Mikkelson, T., Heckl, D., Ebert, B L., Root, D E., Doench, J G.,        Zhang, F. Science December 12. (2013). [Epub ahead of print];    -   Crystal structure of cas9 in complex with guide RNA and target        DNA. Nishimasu, H., Ran, F A., Hsu, P D., Konermann, S.,        Shehata, S I., Dohmae, N., Ishitani, R., Zhang, F., Nureki, O.        Cell February 27, 156(5):935-49 (2014);    -   Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian        cells. Wu X., Scott D A., Kriz A J., Chiu A C., Hsu P D., Dadon        D B., Cheng A W., Trevino A E., Konermann S., Chen S., Jaenisch        R., Zhang F., Sharp P A. Nat Biotechnol. April 20. doi:        10.1038/nbt.2889 (2014);    -   CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling.        Platt R J, Chen S, Zhou Y, Yim M J, Swiech L, Kempton H R,        Dahlman J E, Parnas 0, Eisenhaure™, Jovanovic M, Graham D B,        Jhunjhunwala S, Heidenreich M, Xavier R J, Langer R, Anderson D        G, Hacohen N, Regev A, Feng G, Sharp P A, Zhang F. Cell 159(2):        440-455 DOI: 10.1016[j.cell.2014.09.014(2014);    -   Development and Applications of CRISPR-Cas9 for Genome        Engineering, Hsu P D, Lander E S, Zhang F., Cell. June 5;        157(6):1262-78 (2014).    -   Genetic screens in human cells using the CRISPR/Cas9 system,        Wang T, Wei J J, Sabatini D M, Lander E S., Science. January 3;        343(6166): 80-84. doi:10.1126/science.1246981 (2014);    -   Rational design of highly active sgRNAs for CRISPR-Cas9-mediated        gene inactivation, Doench J G, Hartenian E, Graham D B, Tothova        Z, Hegde M, Smith I, Sullender M, Ebert B L, Xavier R J, Root D        E., (published online 3 Sep. 2014) Nat Biotechnol. December;        32(12):1262-7 (2014);    -   In vivo interrogation of gene function in the mammalian brain        using CRISPR-Cas9, Swiech L, Heidenreich M, Banerjee A, Habib N,        Li Y, Trombetta J, Sur M, Zhang F., (published online 19        Oct. 2014) Nat Biotechnol. January; 33(1):102-6 (2015);    -   Genome-scale transcriptional activation by an engineered        CRISPR-Cas9 complex, Konermann S, Brigham M D, Trevino A E,        Joung J, Abudayyeh O O, Barcena C, Hsu P D, Habib N, Gootenberg        J S, Nishimasu H, Nureki 0, Zhang F., Nature. Jan 29;        517(7536):583-8 (2015).    -   A split-Cas9 architecture for inducible genome editing and        transcription modulation, Zetsche B, Volz S E, Zhang F.,        (published online 2 Feb. 2015) Nat Biotechnol. February;        33(2):139-42 (2015);    -   Genome-wide CRISPR Screen in a Mouse Model of Tumor Growth and        Metastasis, Chen S, Sanjana N E, Zheng K, Shalem O, Lee K, Shi        X, Scott D A, Song J, Pan J Q, Weissleder R, Lee H, Zhang F,        Sharp P A. Cell 160, 1246-1260, Mar. 12, 2015 (multiplex screen        in mouse), and    -   In vivo genome editing using Staphylococcus aureus Cas9, Ran F        A, Cong L, Yan W X, Scott D A, Gootenberg J S, Kriz A J, Zetsche        B, Shalem O, Wu X, Makarova K S, Koonin E V, Sharp P A, Zhang        F., (published online 1 Apr. 2015), Nature. Aprril 9;        520(7546):186-91 (2015).    -   Shalem et al., “High-throughput functional genomics using        CRISPR-Cas9,” Nature Reviews Genetics 16, 299-311 (May 2015).    -   Xu et al., “Sequence determinants of improved CRISPR sgRNA        design,” Genome Research 25, 1147-1157 (August 2015).    -   Parnas et al., “A Genome-wide CRISPR Screen in Primary Immune        Cells to Dissect Regulatory Networks,” Cell 162, 675-686 (Jul.        30, 2015).    -   Ramanan et al., CRISPR/Cas9 cleavage of viral DNA efficiently        suppresses hepatitis B virus,” Scientific Reports 5:10833. doi:        10.1038/srep10833 (Jun. 2, 2015).    -   Nishimasu et al., Crystal Structure of Staphylococcus aureus        Cas9,” Cell 162, 1113-1126 (Aug. 27, 2015).    -   BCL11A enhancer dissection by Cas9-mediated in situ saturating        mutagenesis, Canver et al., Nature 527(7577):192-7 (Nov.        12, 2015) doi: 10.1038/nature15521. Epub 2015 Sep 16.    -   Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas        System, Zetsche et al., Cell 163, 759-71 (Sep. 25, 2015).    -   Discovery and Functional Characterization of Diverse Class 2        CRISPR-Cas Systems, Shmakov et al., Molecular Cell, 60(3),        385-397 doi: 10.1016/j.molcel.2015.10.008 Epub Oct. 22, 2015.    -   Rationally engineered Cas9 nucleases with improved specificity,        Slaymaker et al., Science 2015 Dec. 1. pii: aad5227. [Epub ahead        of print].        each of which is incorporated herein by reference, may be        considered in the practice of the instant invention, and        discussed briefly below:    -   Cong et al. engineered type II CRISPR-Cas systems for use in        eukaryotic cells based on both Streptococcus thermophilus Cas9        and also Streptococcus pyogenes Cas9 and demonstrated that Cas9        nucleases can be directed by short RNAs to induce precise        cleavage of DNA in human and mouse cells. Their study further        showed that Cas9 as converted into a nicking enzyme can be used        to facilitate homology-directed repair in eukaryotic cells with        minimal mutagenic activity. Additionally, their study        demonstrated that multiple guide sequences can be encoded into a        single CRISPR array to enable simultaneous editing of several at        endogenous genomic loci sites within the mammalian genome,        demonstrating easy programmability and wide applicability of the        RNA-guided nuclease technology. This ability to use RNA to        program sequence specific DNA cleavage in cells defined a new        class of genome engineering tools. These studies further showed        that other CRISPR loci are likely to be transplantable into        mammalian cells and can also mediate mammalian genome cleavage.        Importantly, it can be envisaged that several aspects of the        CRISPR-Cas system can be further improved to increase its        efficiency and versatility.    -   Jiang et al. used the clustered, regularly interspaced, short        palindromic repeats (CRISPR)-associated Cas9 endonuclease        complexed with dual-RNAs to introduce precise mutations in the        genomes of Streptococcus pneumoniae and Escherichia coli. The        approach relied on dual-RNA:Cas9-directed cleavage at the        targeted genomic site to kill unmutated cells and circumvents        the need for selectable markers or counter-selection systems.        The study reported reprogramming dual-RNA:Cas9 specificity by        changing the sequence of short CRISPR RNA (crRNA) to make        single- and multinucleotide changes carried on editing        templates. The study showed that simultaneous use of two crRNAs        enabled multiplex mutagenesis. Furthermore, when the approach        was used in combination with recombineering, in S. pneumoniae,        nearly 100% of cells that were recovered using the described        approach contained the desired mutation, and in E. coli, 65%        that were recovered contained the mutation.    -   Wang et al. (2013) used the CRISPR/Cas system for the one-step        generation of mice carrying mutations in multiple genes which        were traditionally generated in multiple steps by sequential        recombination in embryonic stem cells and/or time-consuming        intercrossing of mice with a single mutation. The CRISPR/Cas        system will greatly accelerate the in vivo study of functionally        redundant genes and of epistatic gene interactions.    -   Konermann et al. (2013) addressed the need in the art for        versatile and robust technologies that enable optical and        chemical modulation of DNA-binding domains based CRISPR Cas9        enzyme and also Transcriptional Activator Like Effectors.    -   Ran et al. (2013-A) described an approach that combined a Cas9        nickase mutant with paired guide RNAs to introduce targeted        double-strand breaks. This addresses the issue of the Cas9        nuclease from the microbial CRISPR-Cas system being targeted to        specific genomic loci by a guide sequence, which can tolerate        certain mismatches to the DNA target and thereby promote        undesired off-target mutagenesis. Because individual nicks in        the genome are repaired with high fidelity, simultaneous nicking        via appropriately offset guide RNAs is required for        double-stranded breaks and extends the number of specifically        recognized bases for target cleavage. The authors demonstrated        that using paired nicking can reduce off-target activity by 50-        to 1,500-fold in cell lines and to facilitate gene knockout in        mouse zygotes without sacrificing on-target cleavage efficiency.        This versatile strategy enables a wide variety of genome editing        applications that require high specificity.    -   Hsu et al. (2013) characterized SpCas9 targeting specificity in        human cells to inform the selection of target sites and avoid        off-target effects. The study evaluated >700 guide RNA variants        and SpCas9-induced indel mutation levels at >100 predicted        genomic off-target loci in 293T and 293FT cells. The authors        that SpCas9 tolerates mismatches between guide RNA and target        DNA at different positions in a sequence-dependent manner,        sensitive to the number, position and distribution of        mismatches. The authors further showed that SpCas9-mediated        cleavage is unaffected by DNA methylation and that the dosage of        SpCas9 and sgRNA can be titrated to minimize off-target        modification. Additionally, to facilitate mammalian genome        engineering applications, the authors reported providing a        web-based software tool to guide the selection and validation of        target sequences as well as off-target analyses.    -   Ran et al. (2013-B) described a set of tools for Cas9-mediated        genome editing via non-homologous end joining (NHEJ) or        homology-directed repair (HDR) in mammalian cells, as well as        generation of modified cell lines for downstream functional        studies. To minimize off-target cleavage, the authors further        described a double-nicking strategy using the Cas9 nickase        mutant with paired guide RNAs. The protocol provided by the        authors experimentally derived guidelines for the selection of        target sites, evaluation of cleavage efficiency and analysis of        off-target activity. The studies showed that beginning with        target design, gene modifications can be achieved within as        little as 1-2 weeks, and modified clonal cell lines can be        derived within 2-3 weeks.    -   Shalem et al. described a new way to interrogate gene function        on a genome-wide scale. Their studies showed that delivery of a        genome-scale CRISPR-Cas9 knockout (GeCKO) library targeted        18,080 genes with 64,751 unique guide sequences enabled both        negative and positive selection screening in human cells. First,        the authors showed use of the GeCKO library to identify genes        essential for cell viability in cancer and pluripotent stem        cells. Next, in a melanoma model, the authors screened for genes        whose loss is involved in resistance to vemurafenib, a        therapeutic that inhibits mutant protein kinase BRAF. Their        studies showed that the highest-ranking candidates included        previously validated genes NF1 and MED12 as well as novel hits        NF2, CUL3, TADA2B, and TADA1. The authors observed a high level        of consistency between independent guide RNAs targeting the same        gene and a high rate of hit confirmation, and thus demonstrated        the promise of genome-scale screening with Cas9.    -   Nishimasu et al. reported the crystal structure of Streptococcus        pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 A°        resolution. The structure revealed a bilobed architecture        composed of target recognition and nuclease lobes, accommodating        the sgRNA:DNA heteroduplex in a positively charged groove at        their interface. Whereas the recognition lobe is essential for        binding sgRNA and DNA, the nuclease lobe contains the HNH and        RuvC nuclease domains, which are properly positioned for        cleavage of the complementary and non-complementary strands of        the target DNA, respectively. The nuclease lobe also contains a        carboxyl-terminal domain responsible for the interaction with        the protospacer adjacent motif (PAM). This high-resolution        structure and accompanying functional analyses have revealed the        molecular mechanism of RNA-guided DNA targeting by Cas9, thus        paving the way for the rational design of new, versatile        genome-editing technologies.    -   Wu et al. mapped genome-wide binding sites of a catalytically        inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with        single guide RNAs (sgRNAs) in mouse embryonic stem cells        (mESCs). The authors showed that each of the four sgRNAs tested        targets dCas9 to between tens and thousands of genomic sites,        frequently characterized by a 5-nucleotide seed region in the        sgRNA and an NGG protospacer adjacent motif (PAM). Chromatin        inaccessibility decreases dCas9 binding to other sites with        matching seed sequences; thus 70% of off-target sites are        associated with genes. The authors showed that targeted        sequencing of 295 dCas9 binding sites in mESCs transfected with        catalytically active Cas9 identified only one site mutated above        background levels. The authors proposed a two-state model for        Cas9 binding and cleavage, in which a seed match triggers        binding but extensive pairing with target DNA is required for        cleavage.    -   Platt et al. established a Cre-dependent Cas9 knockin mouse. The        authors demonstrated in vivo as well as ex vivo genome editing        using adeno-associated virus (AAV)-, lentivirus-, or        particle-mediated delivery of guide RNA in neurons, immune        cells, and endothelial cells.    -   Hsu et al. (2014) is a review article that discusses generally        CRISPR-Cas9 history from yogurt to genome editing, including        genetic screening of cells.    -   Wang et al. (2014) relates to a pooled, loss-of-function genetic        screening approach suitable for both positive and negative        selection that uses a genome-scale lentiviral single guide RNA        (sgRNA) library.    -   Doench et al. created a pool of sgRNAs, tiling across all        possible target sites of a panel of six endogenous mouse and        three endogenous human genes and quantitatively assessed their        ability to produce null alleles of their target gene by antibody        staining and flow cytometry. The authors showed that        optimization of the PAM improved activity and also provided an        on-line tool for designing sgRNAs.    -   Swiech et al. demonstrate that AAV-mediated SpCas9 genome        editing can enable reverse genetic studies of gene function in        the brain.    -   Konermann et al. (2015) discusses the ability to attach multiple        effector domains, e.g., transcriptional activator, functional        and epigenomic regulators at appropriate positions on the guide        such as stem or tetraloop with and without linkers.    -   Zetsche et al. demonstrates that the Cas9 enzyme can be split        into two and hence the assembly of Cas9 for activation can be        controlled.    -   Chen et al. relates to multiplex screening by demonstrating that        a genome-wide in vivo CRISPR-Cas9 screen in mice reveals genes        regulating lung metastasis.    -   Ran et al. (2015) relates to SaCas9 and its ability to edit        genomes and demonstrates that one cannot extrapolate from        biochemical assays. Shalem et al. (2015) described ways in which        catalytically inactive Cas9 (dCas9) fusions are used to        synthetically repress (CRISPRi) or activate (CRISPRa)        expression, showing. advances using Cas9 for genome-scale        screens, including arrayed and pooled screens, knockout        approaches that inactivate genomic loci and strategies that        modulate transcriptional activity.

End Edits

-   -   Shalem et al. (2015) described ways in which catalytically        inactive Cas9 (dCas9) fusions are used to synthetically repress        (CRISPRi) or activate (CRISPRa) expression, showing. advances        using Cas9 for genome-scale screens, including arrayed and        pooled screens, knockout approaches that inactivate genomic loci        and strategies that modulate transcriptional activity.    -   Xu et al. (2015) assessed the DNA sequence features that        contribute to single guide RNA (sgRNA) efficiency in        CRISPR-based screens. The authors explored efficiency of        CRISPR/Cas9 knockout and nucleotide preference at the cleavage        site. The authors also found that the sequence preference for        CRISPRi/a is substantially different from that for CRISPR/Cas9        knockout.    -   Parnas et al. (2015) introduced genome-wide pooled CRISPR-Cas9        libraries into dendritic cells (DCs) to identify genes that        control the induction of tumor necrosis factor (Tnf) by        bacterial lipopolysaccharide (LPS). Known regulators of Tlr4        signaling and previously unknown candidates were identified and        classified into three functional modules with distinct effects        on the canonical responses to LPS. Ramanan et al (2015)        demonstrated cleavage of viral episomal DNA (cccDNA) in infected        cells. The HBV genome exists in the nuclei of infected        hepatocytes as a 3.2 kb double-stranded episomal DNA species        called covalently closed circular DNA (cccDNA), which is a key        component in the HBV life cycle whose replication is not        inhibited by current therapies. The authors showed that sgRNAs        specifically targeting highly conserved regions of HBV robustly        suppresses viral replication and depleted cccDNA.    -   Nishimasu et al. (2015) reported the crystal structures of        SaCas9 in complex with a single guide RNA (sgRNA) and its        double-stranded DNA targets, containing the 5′-TTGAAT-3′ PAM and        the 5′-TTGGGT-3′ PAM. A structural comparison of SaCas9 with        SpCas9 highlighted both structural conservation and divergence,        explaining their distinct PAM specificities and orthologous        sgRNA recognition.    -   Canver et al. (2015) demonstrated a CRISPR-Cas9-based functional        investigation of non-coding genomic elements. The authors we        developed pooled CRISPR-Cas9 guide RNA libraries to perform in        situ saturating mutagenesis of the human and mouse BCL11A        enhancers which revealed critical features of the enhancers.    -   Zetsche et al. (2015) reported characterization of Cpf1, a class        2 CRISPR nuclease from Francisella novicida U112 having features        distinct from Cas9. Cpf1 is a single RNA-guided endonuclease        lacking tracrRNA, utilizes a T-rich protospacer-adjacent motif,        and cleaves DNA via a staggered DNA double-stranded break.    -   Shmakov et al. (2015) reported three distinct Class 2 CRISPR-Cas        systems. Two system CRISPR enzymes (C2c1 and C2c3) contain        RuvC-like endonuclease domains distantly related to Cpf1. Unlike        Cpf1, C2c1 depends on both crRNA and tracrRNA for DNA cleavage.        The third enzyme (C2c2) contains two predicted HEPN RNase        domains and is tracrRNA independent.    -   Slaymaker et al (2015) reported the use of structure-guided        protein engineering to improve the specificity of Streptococcus        pyogenes Cas9 (SpCas9). The authors developed “enhanced        specificity” SpCas9 (eSpCas9) variants which maintained robust        on-target cleavage with reduced off-target effects.

Also, “Dimeric CRISPR RNA-guided FokI nucleases for highly specificgenome editing”, Shengdar Q. Tsai, Nicolas Wyvekens, Cyd Khayter,Jennifer A. Foden, Vishal Thapar, Deepak Reyon, Mathew J. Goodwin,Martin J. Aryee, J. Keith Joung Nature Biotechnology 32(6): 569-77(2014), relates to dimeric RNA-guided FokI Nucleases that recognizeextended sequences and can edit endogenous genes with high efficienciesin human cells.

With respect to general information on CRISPR-Cas Systems, componentsthereof, and delivery of such components, including methods, materials,delivery vehicles, vectors, particles, AAV, and making and usingthereof, including as to amounts and formulations, all useful in thepractice of the instant invention, reference is made to: U.S. Pat. Nos.8,999,641, 8,993,233, 8,945,839, 8,932,814, 8,906,616, 8,895,308,8,889,418, 8,889,356, 8,871,445, 8,865,406, 8,795,965, 8,771,945 and8,697,359; US Patent Publications US 2014-0310830 (U.S. application Ser.No. 14/105,031), US 2014-0287938 A1 (U.S. application Ser. No.14/213,991), US 2014-0273234 A1 (U.S. application Ser. No. 14/293,674),US2014-0273232 A1 (U.S. application Ser. No. 14/290,575), US2014-0273231 (U.S. application Ser. No. 14/259,420), US 2014-0256046 A1(U.S. application Ser. No. 14/226,274), US 2014-0248702 A1 (U.S.application Ser. No. 14/258,458), US 2014-0242700 A1 (U.S. applicationSer. No. 14/222,930), US 2014-0242699 A1 (U.S. application Ser. No.14/183,512), US 2014-0242664 A1 (U.S. application Ser. No. 14/104,990),US 2014-0234972 A1 (U.S. application Ser. No. 14/183,471), US2014-0227787 A1 (U.S. application Ser. No. 14/256,912), US 2014-0189896A1 (U.S. application Ser. No. 14/105,035), US 2014-0186958 (U.S.application Ser. No. 14/105,017), US 2014-0186919 A1 (U.S. applicationSer. No. 14/104,977), US 2014-0186843 A1 (U.S. application Ser. No.14/104,900), US 2014-0179770 A1 (U.S. application Ser. No. 14/104,837)and US 2014-0179006 A1 (U.S. application Ser. No. 14/183,486), US2014-0170753 (U.S. application Ser. No. 14/183,429), US 2015-0184139(U.S. application Ser. No. 14/324,960), Ser. No. 14/054,414; EuropeanPatents EP 2 764 103 (EP13824232.6), EP 2 784 162 (EP14170383.5) and EP2 771 468 (EP13818570.7); and PCT Patent Publications WO 2014/093661(PCT/US2013/074743), WO 2014/093694 (PCT/US2013/074790), WO 2014/093595(PCT/US2013/07461 1), WO 2014/093718 (PCT/US2013/074825), WO 2014/093709(PCT/US2013/074812), WO 2014/093622 (PCT/US2013/074667), WO 2014/093635(PCT/US2013/074691), WO 2014/093655 (PCT/US2013/074736), WO 2014/093712(PCT/US2013/074819), WO 2014/093701 (PCT/US2013/074800), WO 2014/018423(PCT/US2013/051418), WO 2014/204723 (PCT/US2014/041790), WO 2014/204724(PCT/US2014/041800), WO 2014/204725 (PCT/US2014/041803), WO 2014/204726(PCT/US2014/041804), WO 2014/204727 (PCT/US2014/041806), WO 2014/204728(PCT/US2014/041808), WO 2014/204729 (PCT/US2014/041809), WO 2015/089351(PCT/US2014/069897), WO 2015/089354 (PCT/US2014/069902), WO 2015/089364(PCT/US2014/069925), WO 2015/089427 (PCT/US2014/070068), WO 2015/089462(PCT/US2014/070127), WO 2015/089419 (PCT/US2014/070057), WO 2015/089465(PCT/US2014/070135), WO 2015/089486 (PCT/US2014/070175),PCT/US2015/051691, PCT/US2015/051830. Reference is also made to U.S.provisional patent applications 61/758,468; 61/802,174; 61/806,375;61/814,263; 61/819,803 and 61/828,130, filed on Jan. 30, 2013; Mar. 15,2013; Mar. 28, 2013; Apr. 20, 2013; May 6, 2013 and May 28, 2013respectively. Reference is also made to U.S. provisional patentapplication 61/836,123, filed on Jun. 17, 2013. Reference isadditionally made to U.S. provisional patent applications 61/835,931,61/835,936, 61/836,127, 61/836,101, 61/836,080 and 61/835,973, eachfiled Jun. 17, 2013. Further reference is made to U.S. provisionalpatent applications 61/862,468 and 61/862,355 filed on Aug. 5, 2013;61/871,301 filed on Aug. 28, 2013; 61/960,777 filed on Sep. 25, 2013 and61/961,980 filed on Oct. 28, 2013. Reference is yet further made to: PCTPatent applications Nos: PCT/US2014/041803, PCT/US2014/041800,PCT/US2014/041809, PCT/US2014/041804 and PCT/US2014/041806, each filedJun. 10, 2014; PCT/US2014/041808 filed Jun. 11, 2014; andPCT/US2014/62558 filed Oct. 28, 2014, and U.S. Provisional PatentApplications Ser. Nos. 61/915,148, 61/915,150, 61/915,153, 61/915,203,61/915,251, 61/915,301, 61/915,267,61/915,260, and 61/915,397, eachfiled Dec. 12, 2013; 61/757,972 and 61/768,959, filed on Jan. 29, 2013and Feb. 25, 2013; 61/835,936, 61/836,127, 61/836,101, 61/836,080,61/835,973, and 61/835,931, filed Jun. 17, 2013; 62/010,888 and62/010,879, both filed Jun. 11, 2014; 62/010,329 and 62/010,441, eachfiled Jun. 10, 2014; 61/939,228 and 61/939,242, each filed Feb. 12,2014; 61/980,012, filed Apr. 15, 2014; 62/038,358, filed Aug. 17, 2014;62/054,490, 62/055,484, 62/055,460 and 62/055,487, each filed Sep. 25,2014; and 62/069,243, filed Oct. 27, 2014. Reference is made to U.S.provisional patent application 61/930,214 filed on Jan. 22, 2014.

Mention is also made of U.S. application 62/180,709, filed 17 Jun. 2015,PROTECTED GUIDE RNAS (PGRNAS); U.S. application 62/091,455, filed, 12Dec. 2014, PROTECTED GUIDE RNAS (PGRNAS); U.S. application 62/096,708,24 Dec. 2014, PROTECTED GUIDE RNAS (PGRNAS); U.S. application62/091,462, 12 Dec. 2014,DEAD GUIDES FOR CRISPR TRANSCRIPTION FACTORS;U.S. application 62/096,324, 23 Dec. 2014, 62/180,681, 17 Jun. 2015, and62/237,496, 5 Oct. 2015, DEAD GUIDES FOR CRISPR TRANSCRIPTION FACTORS;U.S. application 62/091,456, 12 Dec. 2014 and 62/180,692, 17 Jun. 2015,ESCORTED AND FUNCTIONALIZED GUIDES FOR CRISPR-CAS SYSTEMS; U.S.application 62/091,461, 12 Dec. 2014, DELIVERY, USE AND THERAPEUTICAPPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR GENOMEEDITING AS TO HEMATOPOIETIC STEM CELLS (HSCs); U.S. application62/094,903, 19 Dec 2014, UNBIASED IDENTIFICATION OF DOUBLE-STRAND BREAKSAND GENOMIC REARRANGEMENT BY GENOME-WISE INSERT CAPTURE SEQUENCING; U.S.application 62/096,761, 24 Dec. 2014, ENGINEERING OF SYSTEMS, METHODSAND OPTIMIZED ENZYME AND GUIDE SCAFFOLDS FOR SEQUENCE MANIPULATION; U.S.application 62/098,059, 30 Dec. 2014 and 62/181,667, 18 Jun. 2015,RNA-TARGETING SYSTEM; U.S. application 62/096,656, 24 Dec. 2014 and62/181,151, 17 Jun. 2015, CRISPR HAVING OR ASSOCIATED WITHDESTABILIZATION DOMAINS; U.S. application 62/096,697, 24 Dec. 2014,CRISPR HAVING OR ASSOCIATED WITH AAV; U.S. application 62/098,158, 30Dec. 2014, ENGINEERED CRISPR COMPLEX INSERTIONAL TARGETING SYSTEMS; U.S.application 62/151,052, 22 Apr. 2015, CELLULAR TARGETING FOREXTRACELLULAR EXOSOMAL REPORTING; U.S. application 62/054,490, 24 Sep.2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CASSYSTEMS AND COMPOSITIONS FOR TARGETING DISORDERS AND DISEASES USINGPARTICLE DELIVERY COMPONENTS; U.S. application 61/939,154, 12 Feb. 2014,SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION WITHOPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S. application 62/055,484, 25Sep. 2014, SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATIONWITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S. application62/087,537, 4 Dec. 2014, SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCEMANIPULATION WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S.application 62/054,651, 24 Sep. 2014, DELIVERY, USE AND THERAPEUTICAPPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR MODELINGCOMPETITION OF MULTIPLE CANCER MUTATIONS IN VIVO; U.S. application62/067,886, 23 Oct. 2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OFTHE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR MODELING COMPETITION OFMULTIPLE CANCER MUTATIONS IN VIVO; U.S. application 62/054,675, 24 Sep.2014 and 62/181,002, 17 Jun. 2015, DELIVERY, USE AND THERAPEUTICAPPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS IN NEURONALCELLS/TISSUES; U.S. application 62/054,528, 24 Sep. 2014, DELIVERY, USEAND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONSIN IMMUNE DISEASES OR DISORDERS; U.S. application 62/055,454, 25 Sep.2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CASSYSTEMS AND COMPOSITIONS FOR TARGETING DISORDERS AND DISEASES USING CELLPENETRATION PEPTIDES (CPP); U.S. application 62/055,460, 25 Sep. 2014,MULTIFUNCTIONAL-CRISPR COMPLEXES AND/OR OPTIMIZED ENZYME LINKEDFUNCTIONAL-CRISPR COMPLEXES; U.S. application 62/087,475, 4 Dec. 2014,FUNCTIONAL SCREENING WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S.application 62/055,487, 25 Sep. 2014, FUNCTIONAL SCREENING WITHOPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S. application 62/087,546, 4Dec. 2014 and 62/181,687, 18 Jun. 2015, MULTIFUNCTIONAL CRISPR COMPLEXESAND/OR OPTIMIZED ENZYME LINKED FUNCTIONAL-CRISPR COMPLEXES; and U.S.application 62/098,285, 30 Dec. 2014, CRISPR MEDIATED IN VIVO MODELINGAND GENETIC SCREENING OF TUMOR GROWTH AND METASTASIS.

Mention is made of U.S. applications 62/181,659, 18 Jun. 2015 and62/207,318, 19 Aug. 2015, ENGINEERING AND OPTIMIZATION OF SYSTEMS,METHODS, ENZYME AND GUIDE SCAFFOLDS OF CAS9 ORTHOLOGS AND VARIANTS FORSEQUENCE MANIPULATION. Mention is made of U.S. application 62/181,675,18 Jun. 2015, and filed 22 Oct. 2015, NOVEL CRISPR ENZYMES AND SYSTEMS,U.S. application 62/232,067, 24 Sep. 2015, U.S. application 62/205,733,16 Aug. 2015, U.S. application 62/201,542, 5 Aug. 2015, U.S. application62/193,507, 16 Jul. 2015, and U.S. application 62/181,739, 18 Jun. 2015,each entitled NOVEL CRISPR ENZYMES AND SYSTEMS and of U.S. application62/245,270, 22 Oct. 2015, NOVEL CRISPR ENZYMES AND SYSTEMS. Mention isalso made of U.S. application 61/939,256, 12 Feb. 2014, and WO2015/089473 (PCT/US2014/070152), 12 Dec. 2014, each entitled ENGINEERINGOF SYSTEMS, METHODS AND OPTIMIZED GUIDE COMPOSITIONS WITH NEWARCHITECTURES FOR SEQUENCE MANIPULATION. Mention is also made ofPCT/US2015/045504, 15 Aug. 2015, U.S. application 62/180,699, 17 Jun.2015, and U.S. application 62/038,358, 17 Aug. 2014, each entitledGENOME EDITING USING CAS9 NICKASES.

Each of these patents, patent publications, and applications, and alldocuments cited therein or during their prosecution (“appln citeddocuments”) and all documents cited or referenced in the appln citeddocuments, together with any instructions, descriptions, productspecifications, and product sheets for any products mentioned therein orin any document therein and incorporated by reference herein, are herebyincorporated herein by reference, and may be employed in the practice ofthe invention. All documents (e.g., these patents, patent publicationsand applications and the appln cited documents) are incorporated hereinby reference to the same extent as if each individual document wasspecifically and individually indicated to be incorporated by reference.

In addition, mention is made of PCT application PCT/US14/70057, entitled“DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMSAND COMPOSITIONS FOR TARGETING DISORDERS AND DISEASES USING PARTICLEDELIVERY COMPONENTS (claiming priority from one or more or all of USprovisional patent applications: 62/054,490, filed Sep. 24, 2014;62/010,441, filed Jun. 10, 2014; and 61/915,118, 61/915,215 and61/915,148, each filed on Dec. 12, 2013) (“the Particle Delivery PCT”),incorporated herein by reference, with respect to a method of preparingan sgRNA-and-Cas9 protein containing particle comprising admixing amixture comprising an sgRNA and Cas9 protein (and optionally HDRtemplate) with a mixture comprising or consisting essentially of orconsisting of surfactant, phospholipid, biodegradable polymer,lipoprotein and alcohol; and particles from such a process. For example,wherein Cas9 protein and sgRNA were mixed together at a suitable, e.g.,3:1 to 1:3 or 2:1 to 1:2 or 1:1 molar ratio, at a suitable temperature,e.g., 15-30C, e.g., 20-25C, e.g., room temperature, for a suitable time,e.g., 15-45, such as 30 minutes, advantageously in sterile, nucleasefree buffer, e.g., 1×PBS. Separately, particle components such as orcomprising: a surfactant, e.g., cationic lipid, e.g.,1,2-dioleoyl-3-trimethylammonium-propane (DOTAP); phospholipid, e.g.,dimyristoylphosphatidylcholine (DMPC); biodegradable polymer, such as anethylene-glycol polymer or PEG, and a lipoprotein, such as a low-densitylipoprotein, e.g., cholesterol were dissolved in an alcohol,advantageously a C₁₋₆ alkyl alcohol, such as methanol, ethanol,isopropanol, e.g., 100% ethanol. The two solutions were mixed togetherto form particles containing the Cas9-sgRNA complexes. Accordingly,sgRNA may be pre-complexed with the Cas9 protein, before formulating theentire complex in a particle. Formulations may be made with a differentmolar ratio of different components known to promote delivery of nucleicacids into cells (e.g. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP),1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC), polyethyleneglycol (PEG), and cholesterol) For example DOTAP: DMPC: PEG: CholesterolMolar Ratios may be DOTAP 100, DMPC 0, PEG 0, Cholesterol 0; or DOTAP90, DMPC 0, PEG 10, Cholesterol 0; or DOTAP 90, DMPC 0, PEG 5,Cholesterol 5. DOTAP 100, DMPC 0, PEG 0, Cholesterol 0. That applicationaccordingly comprehends admixing sgRNA, Cas9 protein and components thatform a particle; as well as particles from such admixing. Aspects of theinstant invention can involve particles; for example, particles using aprocess analogous to that of the Particle Delivery PCT, e.g., byadmixing a mixture comprising sgRNA and/or Cas9 as in the instantinvention and components that form a particle, e.g., as in the ParticleDelivery PCT, to form a particle and particles from such admixing (or,of course, other particles involving sgRNA and/or Cas9 as in the instantinvention).

The present invention will be further illustrated in the followingExamples which are given for illustration purposes only and are notintended to limit the invention in any way.

EXAMPLES Example 1: Origin and Evolution of Adaptive Immunity Systems

Classification and annotation of CRISPR-Cas systems in archaeal andbacterial genomes. The CRISPR-Cas loci has more than 50 gene familiesand there is no strictly universal genes, fast evolution, extremediversity of loci architecture. Therefore, no single tree feasible and amulti-pronged approach is needed. So far, there is comprehensive casgene identification of 395 profiles for 93 Cas proteins. Classificationincludes signature gene profiles plus signatures of locus architecture.

A new classification of CRISPR-Cas systems is proposed in FIG. 1. Class1 includes multisubunit crRNA-effector complexes (Cascade) and Class 2includes Single-subunit crRNA-effector complexes (Cas9-like). FIG. 2provides a molecular organization of CRISPR-Cas. FIG. 3A-3D providesstructures of Type I and III effector complexes: commonarchitecture/common ancestry despite extensive sequence divergence. FIG.4 shows CRISPR-Cas as a RNA recognition motif (RRM)-centered system.FIG. 5 shows Cas1 phylogeny where recombination of adaptation andcrRNA-effector modules show a major aspect of CRISPR-Cas evolution. FIG.6 shows a CRISPR-Cas census, specifically a distribution of CRISPR-Castypes/subtypes among archaea and bacteria.

Cas1 is not always linked to CRISPR-Cas systems, therefore it may bepossible that there are two branches of “solo” Cas1 which suggests theremay be differences in function and origin and possible novel mobileelements (see Makarova, Krupovic, Koonin, Frontiers Genet 2014). Thegenome organization of three casposon families may provide some clues.In addition to Cas1 and PolB, casposons incorporate diverse genesincluding various nucleases (Krupovic et al. BMC Biology 2014). Onefamily has protein-primed polymerase, another family has RNA-primedpolymerase. In addition to diverse Euryarchaeota and Thaumarchaeota,casposons found in several bacteria which suggests horizontal mobility.Casposon Cas1 (transposase/integrase) suggests a basal clade in the Cas1phylogeny.

Bacteria and archaea utilize CRISPR for adaptive immunity in prokaryotesand eukaryotes via genome manipulation. Cas 1 provides a ready made toolfor genome manipulation. There are similar mechanisms of integration incasposons and CRISPR, specifically replication-dependent acquisition bycopy/paste not cut-and-paste (Krupovic et al. BMC Biology 2014). Cas1 isa bona fide integrase (Nuftez J K, Lee A S, Engelman A, Doudna J A.Integrase-mediated spacer acquisition during CRISPR-Cas adaptiveimmunity. Nature. 2015 Feb 18). There is similarity between terminalinverted repeats of casposons and CRISPR (Krupovic et al. BMC Biology2014). CRISPR-Cas may have originated from a casposon and an innateimmunity locus (Koonin, Krupovic, Nature Rev Genet, 2015). The evolutionof adaptive immunity systems in prokaryotes and animals may have beenalong parallel courses with transposon integration at innate immunityloci (Koonin, Krupovic, Nature Rev Genet, 2015). RAG1 transposase (thekey enzyme of V(D)J recombination in vertebrates) may have originatedfrom Transib transposons (Kapitonov V V, Jurka J. RAG1 core and V(D)Jrecombination signal sequences were derived from Transib transposons.PLoS Biol. 2005 June; 3(6):e181), however, none of the Transibs encodesRAG2. RAG1 and RAG2 encoding transposons are described in Kapitonov,Koonin, Biol Direct 2015 and Transib transposase phylogeny is presentedin Kapitonov, Koonin, Biol Direct 2015. Defensive DNA elimination inciliates evolved from a PiggyMAc transposon and RNAi, an innate immunesystem (Swart E C, Nowacki M. The eukaryotic way to defend and editgenomes by sRNA-targeted DNA deletion. Ann N Y Acad Sci. 2015).

The relative stability of the classification implies that the mostprevalent variants of CRISPR-Cas systems are already known. However, theexistence of rare, currently unclassifiable variants implies thatadditional types and subtypes remain to be characterized (Makarova etal. 2015. Evolutionary classification of CRISPR-Cas systems and casgenes).

Transposons play a key contribution to the evolution of adaptiveimmunity and other systems involved in DNA manipulation. Class 1CRISPR-Cas originate from transposons but only for an adaptation module.Class 2 CRISPR-Cas have both adaptation and effector functions wheremodules may have evolved from different transposons.

Example 2: New Predicted Class 2 CRISPR-Cas Systems and Evidence oftheir Independent Origins from Transposable Elements

The CRISPR-Cas systems of bacterial and archaeal adaptive immunity showextreme diversity of protein composition and genomic loci architecture.These systems are broadly divided into two classes, Class 1 withmultisubunit effector complexes and Class 2 with single-subunit effectormodules exemplified by the Cas9 protein (FIGS. 1A and 1B). Applicantsdeveloped a simple computational pipeline (FIG. 7) to leverage theexpanding genomic and metagenomic databases along with our currentunderstanding of CRISPR-Cas systems for prediction of putative new Class2 CRISPR-Cas systems. Analysis of the database of complete bacterialgenomes using this pipeline resulted in the identification of three newvariants, each represented in diverse bacteria and containing cas1 andcas2 genes along with a third gene encoding a large protein predicted tofunction as the effector module. In the first of these loci, theputative effector protein (C2c1p) contains a RuvC-like nuclease domainand resembles the previously described Cpf1 protein, the predictedeffector of Type V CRISPR-Cas systems; accordingly, the new putativesystem is classified as subtype V-B. In depth comparison of proteinsequences suggests that the RuvC-containing effector proteins, Cas9,Cpf1 and C2C1p independently evolved from different groups oftransposon-encoded TnpB proteins. The second group of new putativeCRISPR-Cas loci encompasses a large protein containing two highlydiverged HEPN domains with predicted RNAse activity. Given the noveltyof the predicted effector protein, these loci are classified as new TypeVI CRISPR-Cas that is likely to target mRNA. Together, the results ofthis analysis show that Class2 CRISPR-Cas systems evolved on multiple,independent occasions, by combination of diverse Cas1-Cas2-encodingadaptation modules with effector proteins derived from different mobileelements. This route of evolution most likely produced multiple variantsof Class 2 systems that remain to be discovered.

The CRISPR-Cas adaptive immunity systems are present in ˜45% bacterialand 90% archaeal genomes and show extreme diversity of Cas proteincomposition and sequence, and genomic loci architecture. Based on thestructural organization of their crRNA-effector complexes, these systemsare divided into two classes, namely class 1, with multisubunit effectorcomplexes, and class 2, with single subunit effector complexes(Makarova, 2015) (FIGS. 1A and 1B). Class 1 systems are much more commonand diverse than Class 2 systems. Class 1 currently is represented by 12distinct subtypes encoded by numerous archaeal and bacterial genomes,whereas class 2 systems include three subtypes of Type II system and theputative Type V that collectively are found in about 10% of sequencedbacterial genomes (with a single archaeal genome encompassing a putativeType system). Class 2 systems typically contain only three or four genesin the cas operon, namely the cas1-cas2 pair of genes that are involvedin adaptation but not in interference, a single multidomain effectorprotein that is responsible for interference but also contributes to thepre-crRNA processing and adaptation, and often a fourth gene withuncharacterized functions that is dispensable in at least some Type IIsystems. In most cases, a CRISPR array and a gene for a distinct RNAspecies known as tracrRNA (trans-encoded small CRISPR RNA) are adjacentto Class 2 cas operons (Chylinski, 2014). The tracrRNA is partiallyhomologous to the repeats within the respective CRISPR array and isessential for the processing of pre-crRNA that is catalyzed by RNAseIII, a ubiquitous bacterial enzyme that is not associated with theCRISPR-cas loci (Deltcheva, 2011) (Chylinski, 2014; Chylinski, 2013).

The Type II multidomain effector protein Cas9 has been functionally andstructurally characterized in exquisite detail. In different bacteria,Cas9 proteins encompass from about 950 to over 1,600 amino acids, suchas between about 950 and 1,400 amino acids, and contain two nucleasedomains, namely a RuvC-like (RNase H fold) and HNH (McrA-like) nucleases(Makarova, 2011). The crystal structure of Cas9 reveals a bilobedorganization of the protein, with distinct target recognition andnuclease lobes, with the latter accommodating both the RuvC and the HNHdomains (Nishimasu, 2014) (Jinek, 2014). Each of the nuclease domains ofCas9 is required for the cleavage of one of the target DNA strands(Jinek, 2012; Sapranauskas, 2011). Recently, Cas9 has been shown tocontribute to all three stages of the CRISPR response, that is not onlytarget DNA cleavage (interference) but also adaptation and pre-crRNAprocessing (Jinek, 2012). More specifically, a distinct domain in thenuclease lobe of Cas9 has been shown to recognize and bind theProtospacer-Associated Motif (PAM) in viral DNA during the adaptationstage (Nishimasu, 2014) (Jinek, 2014) (Heler, 2015; Wei, 2015). At thisstage of the CRISPR response, Cas9 forms a complex with Cas1 and Cas2,the two proteins that are involved in spacer acquisition in allCRISPR-Cas systems (Heler, 2015; Wei, 2015).

The Cas9 protein, combined with tracrRNA, has recently become the keytool for the new generation of genome editing and engineering methods(Gasiunas, 2013; Mali, 2013; Sampson, 2014; Cong, 2015). This utility ofCas9 in genome editing hinges on the fact that in Type II CRISPR-Cassystems, unlike other types of CRISPR-Cas systems, all the activitiesrequired for the target DNA recognition and cleavage are assembledwithin a single, albeit large, multidomain protein. This feature of TypeII systems greatly facilitates the design of efficient tools for genomemanipulation. Importantly, not all variants of Cas9 are equal. Most ofthe work so far has been done with Cas9 from Streptococcus pyogenes butother Cas9 species could offer substantial advantages. As a case inpoint, recent experiments with Cas9 from Staphylococcus aureus that isabout 300 amino acids shorter than the S. pyogenes protein have allowedCas9 packaging into the adeno-associated virus vector, resulting in amajor enhancement of CRISPR-Cas utility for genome editing in vivo (Ran,2015).

Type II CRISPR-Cas systems currently are classified into 3 subtypes(II-A, II-B and II-C) (Makarova, 2011) (Fonfara, 2014; Chylinski, 2013;Chylinski, 2014). In addition to the cas1, cas2 and cas9 genes that areshared by all Type II loci, subtype II-A is characterized by an extragene, csn2, that encodes an inactivated ATPase (Nam, 2011; Koo, 2012;Lee, 2012) that plays a still poorly characterized role in spaceracquisition (Barrangou, 2007; Arslan, 2013) (Heler, 2015). Subtype II-Bsystems lack csn2 but instead contains the cas4 gene that is otherwisetypical of Type I systems and encodes a recB family 5′-3′ exonucleasethat contributes to spacer acquisition by generating recombinogenic DNAends (Zhang, 2012) (Lemak, 2013; Lemak, 2014). The cas1 and cas2 genesof subtype II-B are most closely related to the respective proteins ofType I CRISPR-Cas systems which implies a recombinant origin of thisType II subtype (Chylinski, 2014).

Subtype II-C CRISPR-Cas systems are the minimal variety that consistsonly of the cas1, cas2 and cas9 genes (Chylinski, 2013; Koonin, 2013;Chylinski, 2014). Notably, however, it has been shown that inCampylobacter jejuni spacer acquisition by the Type II-C systemsrequires the participation of Cas4 encoded by a bacteriophage (Hooton,2014). Another distinct feature of subtype II-C is the formation of someof the crRNAs by transcription involves transcription from internalalternative promoters as opposed to processing observed in all otherexperimentally characterized CRISPR-Cas systems (Zhang, 2013).

Recently, the existence of Type V CRISPR-Cas systems has been predictedby comparative analysis of bacterial genomes. These putative novelCRISPR-Cas systems are represented in several bacterial genomes, inparticular those from the genus Francisella and one archaeon,Methanomethylophilus alvus (Vestergaard, 2014). All putative Type V lociencompass cas1, cas2, a distinct gene denoted cpf1 and a CRISPR array(Schunder, 2013) (Makarova, 2015). Cpf1 is a large protein (about 1300amino acids) that contains a RuvC-like nuclease domain homologous to thecorresponding domain of Cas9 along with a counterpart to thecharacteristic arginine-rich cluster of Cas9. However, Cpf1 lacks theHNH nuclease domain that is present in all Cas9 proteins, and theRuvC-like domain is contiguous in the Cpf1 sequence, in contrast to Cas9where it contains long inserts including the HNH domain (Chylinski,2014; Makarova, 2015). These major differences in the domainarchitectures of Cas9 and Cpf1 suggest that the Cpf1-containing systemsshould be classified as a new type. The composition of the putative TypeV systems implies that Cpf1 is a single-subunit effector complex, andaccordingly, these systems are assigned to Class 2 CRISPR-Cas. Some ofthe putative Type V loci encode Cas4 and accordingly resemble subtypeII-B loci, whereas others lack Cas4 and thus are analogous to subtypeII-C.

It has been shown that the closest homologs of Cas9 and Cpf1 proteinsare TnpB proteins that are encoded in IS605 family transposons andcontain the RuvC-like nuclease domain as well as a Zn-finger that has acounterpart in Cpf1. In addition, homologs of TnpB have been identifiedthat contain a HNH domain inserted into the RuvC-like domain and showhigh sequence similarity to Cas9. The role of TnpB in transposonsremains uncertain as it has been shown that this protein is not requiredfor transposition.

Given the homology of Cas9 and Cpf1 to transposon-encoded proteins,Applicants hypothesized that Class 2 CRISPR-Cas systems could haveevolved on multiple occasions as a result of recombination between atransposon and a cas1-cas2 locus. Accordingly, Applicants devised asimple computational strategy to identify genomic loci that could becandidates for novel variants of Class 2. Here Applicants describe thefirst application of this approach that resulted in the identificationof three groups of such candidates two of which appear to be distinctsubtypes of Type V whereas the third one seems to qualify at Type VI.The new variants of Class2 CRISPR-Cas systems are of obvious interest aspotential tools for genome editing and expression regulation.

Database search strategy for detection of candidate novel Class 2CRISPR-Cas loci. Applicants implemented a straightforward computationalapproach to identify candidate novel Class 2 CRISPR-Cas systems (FIG. 7.Pipeline). Because the vast majority of the CRISPR-Cas loci encompass acas1 gene (Makarova, 2011; Makarova, 2015) and the Cas1 sequence is themost highly conserved one among all Cas proteins (Takeuchi, 2012),Applicants reasoned that cas1 is the best possible anchor to identifycandidate new loci using translating PSI-BLAST search with Cas1profiles. After detecting all contigs encoding Cas1 by searching the WGS(whole genome shotgun) and NT (nucleotide) databases at the NCBI, theprotein-coding genes were predicted using GenemarkS within the 20 KBregions upstream and downstream of the cas1 gene. These predicted geneswere annotated using the NCBI Conserved Domain Database (CDD) and Casprotein-specific profiles (Makarova et al., 2015, Nat Rev Microbiol.2015, doi: 10.1038/nrmicro3569), and CRISPR arrays were predicted usingthe PILER-CR program. This procedure provided for assignment of thedetected CRISPR-Cas loci to the known subtypes. Partial and/orunclassified candidate CRISPR-Cas loci containing large (>500 aa)proteins were selected as candidates for novel Class 2 systems given thecharacteristic presence of such large single-subunit effector proteinsin Types II and V systems (Cas9 and Cpf1, respectively). All 63candidate loci detected using these criteria (listed in the Table setforth in FIG. 40A-D) were analyzed on a case by case basis usingPSI-BLAST and HHpred. The protein sequences encoded in the candidateloci were further used as queries to search metagenomic databases foradditional homologs, and long contigs detected in these searches wereanalyzed as indicated above. This analysis pipeline yielded a total of53 novel loci (some of the originally identified 63 candidate loci werediscarded as spurious whereas several incomplete loci that lacked cas1were added) with characteristic features of Class 2 CRISPR-Cas systemsthat could be classified into three distinct groups of loci based on thenature of the predicted effector proteins (see FIGS. 8A and 8B;

FIGS. 9, 14 and 15; and FIGS. 41A-B, 42A-B, and 43A-B). Althoughbacteriophages infecting bacteria that harbor the newly discovered class2 CRISPR-Cas systems are virtually unknown, for each of these systems,we detected spacers that matched phages or predicted prophages.

Using the computational strategy, the Applicants realised three newClass 2 CRISPR-Cas systems, namely C2c1 and C2c3, which are classifiedas subtypes of the previously described putative type V, and C2c2, whichthe Applicants assign to a new putative type VI on the strength of thepresence of a novel predicted effector protein. The Applicants presentmultiple lines of evidence that these loci encode functional CRISPR-Cassystems. On the comparative genomic side, we identified phage-specificspacers for each of the three putative novel systems and also showedthat the sets of spacers are completely different in closely relatedbacterial genomes suggestive of active, functioning immunity. Many ofthese new systems occur in bacterial genomes that encompass no otherCRISPR-Cas loci, suggesting that type V and type VI systems can functionautonomously. Furthermore, even when other CRISPR-Cas systems wereidentified in the same genomes, the associated repeat structures wereclearly distinct from those in types V and VI, suggestive of independentfunctionality.

Putative type V-B system. The first group of candidate loci,provisionally denoted C2c1 (Class 2 candidate 1), is represented inbacterial genomes from four major taxa, including Bacilli,Verrucomicrobia, alpha-proteobacteria and delta-proteobacteria (FIG.8A-B “Organization of complete loci of Class 2 systems”; FIG. 41A-B).All C2c1 loci encode a Cas1-Cas4 fusion, Cas2, and the large proteinthat Applicants denote C2c1p, and typically, are adjacent to a CRISPRarray (FIG. 9, C2c1 neighborhoods; FIG. 41A-B). In the phylogenetic treeof Cas1, the respective Cas1 proteins cluster with Type I-U system(FIGS. 10A and 10B, FIG. 10C-1-W, Cas1 tree), the only one in which theCas1-Cas4 fusion is found. The lengths of the C2c1p proteins identifiedherein range from about 1100 to about 1500 amino acids, for example mayconsist of approximately 1200 amino acids, and HHpred search detectedsignificant similarity between the C-terminal portion of the C2c1pproteins and a subset of TnpB proteins encoded in transposons of theIS605 family (FIGS. 13A-1-A-2 and 13C-1-C-2). In contrast, nosignificant similarity was detected between C2c1p and Cas9 or Cpf1 thatare similar to other groups of TnpB proteins (Chylinski, 2014)(Makarova, 2015; Makarova, 2015). Thus, the domain architecture of C2c1pis similar to that of Cpf1 and distinct from that of Cas9 (FIG.13A-1-A-2) although all three Cas proteins seem to have evolved from theTnpB family (FIG. 11 “Domain organization of class 2 families”; FIG.13A-1-A-2). The N-terminal region of C2c1p shows no significantsimilarity to other proteins. Secondary structure prediction indicatesthat this region adopts mostly alpha-helical conformation. The twosegments of similarity with TnpB encompass the three catalytic motifs ofthe RuvC-like nuclease, with the diagnostic D.E.D signature of catalyticamino acid residues (Aravind et al., 2000, Nucleic Acids Res, vol. 28,3417-3432) (FIG. 12-1-2, “TnpB homology regions in Class 2 proteins”;FIG. 13A-1-A-2); the region corresponding to the bridge helix (alsoknown as arginine-rich cluster) that in Cas9 protein is involved incrRNA-binding; and a small region that appears to be the counterpart tothe Zn finger of TnpB (however, the Zn-binding cysteine residues aremissing in the majority of C2c1 proteins indicating that such proteinsdo not bind zinc; moreover, C2c1 contain multiple insertions anddeletions in this region suggestive of functional divergence (FIG.13A-1-A-2, FIG. 13D-1-H-2, FIG. 13I-1-I-4). The conservation of thecatalytic residues (FIG. 13A-1-A-2) strongly suggests that the RuvChomology domains of all these proteins are active nucleases. TheN-terminal regions of C2c1 show no significant similarity to any knownproteins. Secondary structure predictions indicate that the N-terminalregions of C2c1 proteins adopt a mixed/O conformation (FIG. 13D-1-H-2,FIG. 13I-1-I-4). The similarity of the domain architectures of C2c1p andCpf1 suggests that the C2c1 loci are best classified as Subtype V-B inwhich case the Cpf1-encoding loci become Subtype V-A.

Despite similarity of cas1 genes associated with this system, the CRISPRrepeats in the respective arrays are highly heterogeneous although allof them are 36-37 bp long and can be classified as unstructured (foldingenergy, AG, is −0.5-4.5 kcal/mole whereas highly palindromic CRISPR haveAG below −7 kcal/mole). According to the CRISPRmap (Lange, 2013)classification scheme, several of the Subtype V-B repeats share somesequence or structural similarity with Type II repeats (FIG. 41A-M-2).However, most of the repeats could not be classified into the knownsequence or structure families and were variously assigned to 4 of the 6superclasses (FIG. 41A-M-2).

Considering the possibility that the putative Subtype V-B CRISPR-Cassystems are mechanistically analogous to Type II systems, Applicantsattempted to identify the tracrRNA in the respective genomic loci.

Comparison of the spacers from the Type V-B CRISPR arrays to thenon-redundant nucleotide sequence database identified several matches tovarious bacterial genomes. In particular, one of the spacers fromAlicyclobacillus acidoterrestris and one of the spacers fromBrevibacillus agri matched uncharacterized genes within predictedprophages integrated into the respective bacterial genomes (FIG. 41A-L).

Putative type VI systems. The second group of candidate CRISPR-Cas loci,denoted C2c2 (Class 2 candidate 2), was identified in genomes from 5major bacterial taxa, including alpha-proteobacteria, Bacilli,Clostridia, Fusobacteria and Bacteroidetes (FIG. 8A-B “Organization ofcomplete loci of Class 2 systems”; FIG. 42A-B).A number of C2c2 lociencompass cas1 and cas2 genes, along with a large protein (C2c2p) thatshows no sequence similarity to C2c1, Cpf1, or Cas9, and a CRISPR array;however, unlike C2c1, C2c2p is often encoded next to a CRISPR array butnot cas1-cas2 (FIG. 15, C2c2 neighborhoods; FIG. 42A-B). Although underour computational strategy, the originally identified C2c2 lociencompassed the cas1 and cas2 genes, subsequent searches showed that themajority of such loci may consist only of the c2c2 gene and a CRISPRarray. Such apparently incomplete loci could either encode defectiveCRISPR-Cas systems or might function with the adaptation module encodedelsewhere in the genome, as has been observed for some type III systems(Majumdar et al., 2015, RNA, vol. 21, 1147-1158). In the phylogenetictree of Cas1, the Cas1 proteins from the C2c2 loci are distributed amongtwo clades. The first clade includes Cas1 from Clostridia and is locatedwithin the Type II subtree along with a small Type III-A branch (FIGS.10A and 10B, FIG. 10C-1-W, Cas1 tree). The second clade consists of Cas1proteins from C2c2 loci of Leptotrichia and is lodged inside a mixedbranch that mostly contains Cas1 proteins from Type III-A CRISPR-Cassystems. Database searches using HHpred and PSI-BLAST detected nosequence similarity between C2c2p and other proteins. However,inspection of multiple alignments of C2c2p protein sequences led to theidentification of two strictly conserved RxxxxH motifs that arecharacteristic of HEPN (Higher Eukaryotes and ProkaryotesNucleotide-binding) domains (Anantharaman et al., 2013, Biol Direct,vol. 8, 15; Grynberg et al., 2003, Trends in biochemical sciences, vol.28, 224-226) (FIG. 11 and FIG. 13B, FIG. 13J-1-N-4). Secondary structurepredictions indicates that these motifs are located within structuralcontexts compatible with the HEPN domain structure as is the overallsecondary structure prediction for the respective portions of C2c2p. TheHEPN domains are small (˜150 aa) alpha helical domains with diversesequences but highly conserved catalytic motifs that have been shown orpredicted to possess RNAse activity and are often associated withvarious defense systems (Anantharaman, 2013) (FIGS. 13B and 16-1-4, HEPNRxxxxH motif in C2c2 family). The sequences of HEPN domains show littleconservation except for the catalytic RxxxxH motif. While the sequencesof the two putative HEPN domains of C2c2 show little similarity to otherHEPN domains except for the catalytic RxxxxH motifs, the domain identityis strongly supported by secondary structure predictions that indicatethat each motif is located within compatible structural contexts (FIG.13B, FIG. 13J-1-N-4). Furthermore, the predicted secondary structure ofthe entire sequence for each putative domain is also consistent with theHEPN fold (FIG. 13J-1-N-4). Thus, it appears likely that C2c2p containstwo active HEPN domains. The HEPN domain is not new to CRISPR-Cassystems as it is often associated with the CARF (CRISPR-AssociatedRossmann Fold) domain in Csm6 and Csx1 proteins that are present in manyType III CRISPR-Cas systems (Makarova, 2014). These proteins do notbelong to either the adaptation modules or effector complexes but arethought to perform some accessory, still uncharacterized functions incognate CRISPR, more particularly they appear to be components of theassociated immunity module that is present in the majority of CRISPR-Cassystems and is implicated in programmed cell death as well as regulatoryfunctions during the CRISPR response (Koonin, 2013; Makarova, 2012;Makarova, 2013). However, C2c2p differs from Csm6 and Csx1 in that thismuch larger protein is the only common protein encoded in the C2c2 loci,except for Cas1 and Cas2. Thus, it appears likely that C2c2p is theeffector of these putative novel CRISPR-Cas systems and the HEPN domainsare the catalytic moieties thereof. Outside of the predicted HEPNdomains, the C2c2p sequence showed no detectable similarity to otherproteins and is predicted to adopt a mixed alpha/beta secondarystructure without discernible similarity to any known protein folds(FIG. 13J-1-N-4).

The CRISPR arrays in the C2c2 loci are highly heterogeneous, with thelength of 35 to 39 bp, and unstructured (folding energy of −0.9 to 4.7kcal/mole). According to CRISPRmap (Lange, 2013), these CRISPR do notbelong to any of the established structural classes and are assigned to3 of the 6 superclasses. Only the CRISPR from Listeria seeligeri wasassigned to the sequence family 24 that is usually associated with TypeII-C systems (FIG. 42A-L).

Spacer analysis of the C2c2 loci identified one 30 nucleotide regionidentical to a genomic sequence from Listeria weihenstephanensis and twoimperfect hits to bacteriophage genomes, in particular, a spacer fromListeria weihenstephanensis matched the tail gene of a Listeriabacteriophage (FIG. 42A-L).

Given the unique predicted effector complex of C2c2, these systems seemto qualify as a putative Type VI CRISPR-Cas. Furthermore, taking intoaccount that all experimentally characterized and enzymatically activeHEPN domains are RNAses, Type VI systems are likely to act at the levelof RNA, such as mRNA.

Putative type V-C systems. The third group of candidate loci includessolely metagenomic sequences and thus could not be assigned to specifictaxa. These loci encompass only two protein-coding genes that encodeCas1 and a large protein denoted C2c3 (Class 2 candidate 3) (FIG. 8A“Organization of complete loci of Class 2 systems”; FIG. 14, C2c3neighbourhoods, FIG. 43A-B). The C2c3 proteins are in the same sizerange as Cpf1 and C2c1, and similarly contain a TnpB-homologous domainat their C-termini which, unlike the respective domain of C2c1, showed alimited but significant similarity to Cpf1 (FIGS. 13A-1-A-2 and13C-1-C-2). The TnpB homology regions of C2c3 contain the threecatalytic motifs of the RuvC-like nuclease, with the diagnostic D.E.Dtriad of catalytic amino acid residues (Aravind et al., 2000, supra),the region corresponding to the bridge helix (also known as thearginine-rich cluster), which is involved in crRNA-binding in Cas9, anda small region that appears to be the counterpart to the Zn finger ofTnpB (the Zn-binding cysteine residues are conserved in C2c3). Theconservation of the catalytic residues strongly suggests that the RuvChomology domains of all these proteins are active nucleases. TheN-terminal regions of C2c1 and C2c3 show no significant similarity toeach other or any known proteins. Secondary structure predictionsindicate that the N-terminal regions of C2c3 proteins adopt a mixed/Oconformation. Thus, the overall domain architectures of C2c1 and C2c3,and in particular the organization of the RuvC domain, are similar tothat of Cpf1 but distinct from that of Cas9. This suggests that the C2c1and C2c3 loci are best classified as subtypes V-B (see above) and V-C,respectively, with Cpf1-encoding loci now designated subtype V-A.

Among the c2c3 loci, only one contains a CRISPR array with unusuallyshort, 17-18 nt spacers. The repeats in this array are 25 bp long andappear to be unstructured with folding energy of ˜1.6 kcal/mol (FIG.43A-F).

Spacers from the only C2c3 contig containing a CRISPR array are tooshort to produce statistically significant hits. Nevertheless, severalmatches to sequences from predicted prophages were identified (FIG.43A-F).

The subsets of the TnpB proteins with significant similarity to the oneknown (Cas9) and three herein disclosed putative Class 2 effectors(Cpf1, C2c1 and C2c3) did not overlap (FIGS. 13A-1-A-2 and 13C-1-C-2).Although the sequence divergence among the TnpB-like domains is too highto allow reliable phylogenetic analysis, these findings suggest that thefour currently identified large effector proteins of Class 2, Cas9,Cpf1, C2c1 and C2c3, have evolved independently from genes of distincttransposable elements.

Although the majority of spacers in the new CRISPR-Cas loci describedherein were not significantly similar to any available sequences, theexistence of spacers matching phage genomes implies that these loci mayencode active, functional adaptive immunity systems. The small fractionof phage-specific spacers is typical of CRISPR-Cas systems and is mostlikely indicative of their dynamic evolution and the small fraction ofvirus diversity that is represented in the current sequence databases.This interpretation is compatible with the observation that closelyrelated bacterial strains encoding homologous CRISPR-Cas loci typicallycontain unrelated collections of spacers, as exemplified by the C2c2loci from Listeria weihenstephanensis and Listeria newyorkensis (FIG.45A-C).

Applicants applied a simple, straightforward computational strategy topredict new Class 2 CRISPR-cas systems. The previously described class 2systems, namely Type II and the putative Type V, consisted of the cas1and cas2 genes (and in some cases also cas4) comprising the adaptationmodule and a single large protein that comprises the effector module.Therefore, Applicants surmised that any genomic locus containing cas1and a large protein could be a potential candidate for a novel Class 2system that merits detailed investigation. Such analysis using sensitivemethods for protein sequence comparison led to the identification ofthree strong candidates two of which are distinct subtypes of thepreviously described putative Type V (subtypes V-B and V-C) whereas thethird one qualifies as a new putative Type VI, on the strength of thepresence of a novel predicted effector protein. Many of these newsystems occur in bacterial genomes that encompass no other CRISPR-Casloci (FIG. 44A-E-2) suggesting that Type V and Type VI systems canfunction autonomously. The herein disclosed candidate loci werevalidated through functional assays which revealed the expression andprocessing of the respective CRISPR arrays, yielding mature crRNAs,identification of putative tracrRNA (where present), demonstration ofinterference when expressed in E. coli, determination of the protospaceradjacent motif (PAM), and interrogation of the minimal componentsnecessary for lysate cleavage.

Type V systems encode predicted effector proteins that resemble Cas9 intheir overall domain architecture, but in contrast to Cas9, theRuvC-like domains of Cpf1, C2c1 and C2c3 are contiguous in the proteinsequence, lacking the inserts characteristic of Cas9, particularly theHNH nuclease domain. The presence of one instead of two nuclease domainsindicates that type V effector proteins mechanistically differ from Cas9in which the HNH and RuvC domains are responsible for the cleavage ofthe complementary and non-complementary strands of the target DNA,respectively (Chen et al., 2014, The Journal of biological chemistry,vol. 289, 13284-13294; Gasiunas et al., 2012, Proceedings of theNational Academy of Sciences of the United States of America, vol. 109,E2579-2586; Jinek et al., 2012, Science, vol. 337, 816-821). Thepredicted type V effector proteins might form dimers in which the twoRuvC-like domains would cleave the opposite strands of the targetmolecule.

The putative type VI CRISPR-Cas systems seem to rely on a novel effectorprotein that contains two predicted HEPN domains that, similar to thepreviously characterized HEPN domains, could possess RNAse activity,suggesting that type VI systems might target and cleave mRNA.Previously, mRNA targeting has been reported for certain type IIICRISPR-Cas systems (Hale et al., 2014, Genes Dev, vol. 28, 2432-2443;Hale et al., 2009, Cell, vol. 139, 945-956; Peng et al., 2015, Nucleicacids research, vol. 43, 406-417). An alternative possibility is thatC2c2 is the first DNAse in the HEPN superfamily, perhaps with the twoHEPN domains each cleaving one DNA strand. Thus, it might be possible todevelop C2c1 and C2c2 into genome editing tools with different classesof targets.

To validate the functionality of these Class2 CRISPR-Cas systems, theApplicants showed that two C2c1 CRISPR arrays are expressed, processedinto mature crRNAs, and capable of interference when expressed in E.coli. These experiments revealed several characteristics of the C2c1locus including: (i) a 5′ processed DR on the crRNA, (ii) a 5′ PAM, and(iii) the presence of a short RNA with repeat-anti-repeat homology tothe processed 5′ DR, i.e., a putative tracrRNA. The discovery of a 5′processed DR and 5′ PAM supports the scenario in which C2c1 is derivedfrom Class 1 systems because these systems show evidence of 5′ repeatprocessing (type I and III) and a 5′ PAM (type I) (Mojica et al., 2009,Microbiology, vol. 155, 733-740; Makarova et al., 2011, Nat RevMicrobioln vol. 9, 467-477). Notably, the AT-rich PAM identified herefor C2c1 is in contrast to the GC-rich PAMs of the otherwell-characterized Class 2 system (type II). For C2c1 lociexperimentally characterized here, the Applicants identified crRNAs thatare processed to a length that preserves the binding and co-folding withputative tracrRNAs, suggesting that tracrRNAs may be involved in andpossibly required for complex formation. We then used expression of C2c1in a human cell culture to experimentally test that under thosecircumstances a tracrRNA was involved in and necessary for the in vitrocleavage of target DNA by the particular C2c1 nuclease tested.

The Applicants also showed that when the C2c2 locus from L. seeligeri isexpressed in E. coli, it is processed into crRNAs with a 29-nt 5′ DR;similar results were obtained for the C2c2 locus of L. shahii. In thiscase, the degenerate repeat is at the beginning of the array, ratherthan at the end, as is typical for most CRISPR arrays, and the array andcas genes are transcribed co-directionally. The Applicants did notdetect the putative tracrRNA in the C2c2 RNA-seq data. However, thepredicted secondary structure of the 29-nt DR shows a stable hairpinhandle which could be potentially important for complex formation withthe C2c2 effector protein.

Combined with the results of previous analyses, (Chylinski, 2014;Makarova, 2011), the identification of the novel Class2 CRISPR-Cassystems reveals the dominant theme in the evolution of Class 2CRISPR-Cas systems. The effector proteins of two of the three typeswithin this class appear to have evolved from the pool of transposableelements that encode TnpB proteins containing the RuvC-like domain. Thesequences of the RuvC-like domains of TnpB and the homologous domains ofthe Class 2 effector proteins are too diverged for reliable phylogeneticanalysis. Nevertheless, for Cas9, the effector protein of Type IIsystems, the specific ancestral group seems to be readily identifiable,namely a family of TnpB-like proteins, particularly abundant inCyanobacteria, that show a relatively high sequence similarity to Cas9and share with it the entire domain architecture, namely the RuvC-likeand HNH nuclease domains and the arginine-rich bridge helix (Chylinski,2014) (FIG. 11, FIGS. 13A-1-A-2 and 13B, “Domain organization of class 2families”; FIG. 12-1-2, FIGS. 13A-1-A-2 and 13B, “TnpB homology regionsin Class 2 proteins”). Unlike Cas9, it was impossible to trace Cpf1,C2c1, and C2c3 to a specific TnpB family; despite the conservation ofall motifs centered at the catalytic residues of the RuvC-likenucleases, these proteins show only a limited similarity to genericprofiles of the TnpB. However, given that C2c1p shows no detectablesequence similarity with Cpf1, that Cpf1, C2c1, and C2c3 containdistinct insertions between the RuvC-motifs and clearly unrelatedN-terminal regions, it appears most likely that Cpf1, C2c1, and C2c3originated independently from different families within the pool ofTnpB-encoding elements (FIG. 13C-1-C-2).

It is intriguing that the TnpB proteins seem to be “predesigned” forutilization in Class 2 CRISPR-Cas effector complexes such that theyapparently have been recruited on multiple different occasions.Conceivably, such utility of TnpB proteins has to do with theirpredicted ability to cut a single-stranded DNA while bound to a RNAmolecule via the R-rich bridge helix that in Cas9 has been shown to bindcrRNA (Jinek, 2014; Nishimasu, 2014; Anders et al., 2014, Nature, vol.513, 569-573). The functions of TnpB in the life cycles of therespective transposons are poorly understood. These proteins are notrequired for transposition, and in one case, a TnpB protein has beenshown to down-regulate transposition (Pasternak, 2013) but theirmechanism of action remains unknown. Experimental study of TnpB islikely to shed light on the mechanistic aspects of the Class 2CRISPR-Cas systems. It should be noted that the mechanisms of Cpf1 andC2c1 could be similar to each other but are bound to substantiallydiffer from that of Cas9 because the former two proteins lack the HNHdomain that in Cas9 is responsible for nicking one of the target DNAstrands (Gasiunas, 2012) (Jinek, 2012) (Chen, 2014). Accordingly,exploitation of Cpf1 and C2c1 might bring additional genome editingpossibilities.

In evolutionary terms, it is striking that Class 2 CRISPR-Cas appear tobe completely derived from different transposable elements given therecent evidence on the likely origin of cas1 genes from a distincttransposon family (Koonin, 2015; Krupovic, 2014). Furthermore, thelikely independent origin of the effector proteins from differentfamilies of TnpB, along with the different phylogenetic affinities ofthe respective cas1 proteins, strongly suggest that Class 2 systems haveevolved on multiple occasions through the combination of variousadaptation modules and transposon-derived nucleases giving rise toeffector proteins. This mode of evolution appears to be the ultimatemanifestation of the modularity that is characteristic of CRISPR-Casevolution (Makarova, 2015), with the implication that additionalcombinations of adaptation and effector module are likely to exist innature.

The putative Type VI CRISPR-Cas systems encompass a predicted noveleffector protein that contains two predicted HEPN domain that are likelyto possess RNAse activity. The HEPN domains are not parts of theeffector complexes in other CRISPR-Cas systems but are involved in avariety of defense functions including a predicted ancillary role invarious CRISPR-Cas systems (Anantharaman, 2013) (Makarova, 2015). Thepresence of the HEPN domains as the catalytic moiety of the predictedeffector module implies that the Type VI systems target and cleave mRNA.Previously, mRNA targeting has been reported for certain Type IIICRISPR-Cas systems (Hale, 2014; Hale, 2009) (Peng, 2015). Although HEPNdomains so far have not been detected in bona fide transposableelements, they are characterized by high horizontal mobility and areintegral to mobile elements such as toxin-antitoxin units (Anantharaman,2013). Thus, the putative Type VI systems seem to fit the generalparadigm of the modular evolution of Class 2 CRISPR-Cas from mobilecomponents, and additional variants and new types are expected to bediscovered by analysis of genomic and metagenomics data. Given that theC2c2 protein is unrelated to the other Class 2 effectors (which allcontain RuvC-like domains, even if distantly related ones), thediscovery of type VI can be considered to corroborate the case for theindependent origins of different Class 2 variants.

In view of the emerging scenario of the evolution of Class 2 systemsfrom mobile elements, it seems instructive to examine the overallevolution of CRISPR-Cas loci and in particular the contributions ofmobile elements to this process (FIG. 53). The ancestral adaptiveimmunity system most likely originated via the insertion of a casposon(a Cas1-encoding transposon) adjacent to a locus that encoded aprimitive innate immunity system. (Koonin and Krupovic, 2015, Naturereviews Genetics, vol. 16, 184-192; Krupovic et al., 2014, BMC Biology,vol. 12, 36). An additional important contribution was the incorporationof a toxin-antitoxin system that delivered the cas2 gene and might haveoccurred either in the ancestral casposon or in the evolving adaptiveimmunity locus (FIG. 53).

Given the extremely wide spread of Class 1 systems in archaea andbacteria and the proliferation of the ancient RRM (RNA RecognitionMotif) domains in them, there seems to be little doubt that theancestral system was of Class 1 (FIG. 53). Most likely, the ancestralarchitecture resembled the extant type III and in that it encompassed anenzymatically active Cas10 protein (Makarova et al., 2011, Biol Direct,vol. 6, 38; Makarova et al., 2013, Biochem Soc Trans, vol. 41,1392-1400). The Cas10 protein is a homolog of family B DNA polymerasesand nucleotide cyclases of the GGDEF (SEQ ID NO: 37) family that showssignificant sequence similarity to these enzymes and retains all thecatalytic amino acid residues (Makarova et al., 2011, Biol Direct, vol.6, 38; Makarova et al., 2006, Biol Direct, vol. 1, 7). Structuralanalysis has confirmed the presence of the polymerase-cyclase-likedomain in Cas10 and additionally revealed a second, degenerate andapparently inactive domain of this family (Khachatryan et al., 2015,Phys Rev Lett, vol. 114, 051801; Shao et al., 2013, Structure, vol. 21,376-384; Zhu and Ye, 2012, FEBS Lett, vol. 586, 939-945). The exactnature of the catalytic activity of Cas10 remains unclear but it hasbeen shown that the catalytic residues of the polymerase-cyclase-likedomain are essential for the target DNA cleavage (Samai et al., 2015,Cell, vol. 161, 1164-1174). The Cas8 proteins present in type ICRISPR-Cas systems are similar in size to Cas10 and occupy equivalentpositions in the effector complexes (Jackson et al., 2014, Science, vol.345, 1473-1479; Jackson and Wiedenheft, 2015, Mol Cell, vol. 58,722-728; Staals et al., 2014, Molecular cell, vol. 56, 518-530),suggestive of an evolutionary relationship between the large subunits ofthe type III and type I effector complexes. More specifically, the Cas8proteins that have highly diverged in sequence between type I subtypescould be catalytically inactive derivatives of Cas10 (Makarova et al.,2011, Biol Direct, vol. 6, 38; Makarova et al., 2015). This scenariosuggests a plausible directionality of evolution, from type III-likeancestral Class 1 system to the type I systems. The divergence of thetype III and type I systems could have been precipitated by theacquisition of the Cas3 helicase by the emerging type I (FIG. 53). Thedifferent types and subtypes of Class 2 then evolved via multiplesubstitutions of the gene block encoding the Class 1 effector complexesvia insertion of transposable elements encoding various nucleases (FIG.53). This particular directionality of evolution follows from theobservation that the adaptation modules of different Class 2 variantsderive from different Class 1 types (FIGS. 10A and 10B).

The Class 2 CRISPR-Cas systems appear to have been completely derivedfrom different mobile elements. Specifically, there seem to have been atleast two (in subtype V-C) but typically, three or, in the case of typeII, even four mobile element contributors: (i) the ancestral casposon,(ii) the toxin-antitoxin module that gave rise to Cas2, (iii) atransposable element, in many cases a TnpB-encoding one, that was theancestor of the Class 2 effector complex, and (iv) in the case of typeII, the HNH nuclease could have been donated to the ancestral transposonby a group I or group II self-splicing intron (Stoddard, 2005, Q RevBiophys, vol. 38, 49-95) (FIG. 53). The putative type V-C loci describedhere encode the ultimate minimalistic CRISPR-Cas system, the onlycurrently identified one that lacks Cas2; conceivably, the highlydiverged subtype V-C Cas1 proteins are capable of forming the adaptationcomplex on their own, without the accessory Cas2 subunit. The multipleoriginations of Class 2 systems from mobile elements present theultimate manifestation of the modularity that is characteristic of theevolution of CRISPR-Cas (Makarova et al., 2015).

The demonstration that different varieties of Class 2 CRISPR-Cas systemsindependently evolved from different transposable elements implies thatadditional variants and new types remain to be identified. Although mostif not all of the new CRISPR-Cas systems are expected to be rare, theycould employ novel strategies and molecular mechanisms and could providea major resource for new, versatile applications in genome engineeringand biotechnology.

Modular evolution is a key feature of CRISPR-Cas systems. This mode ofevolution appears to be most pronounced in Class 2 systems that evolvethrough the combination of adaptation modules from various otherCRISPR-Cas systems with effector proteins that seem to be recruited frommobile elements on multiple independent occasions. Given the extremediversity of mobile elements in bacteria, it appears likely thateffector modules of Class 2 CRISPR-Cas systems are highly diverse aswell. Here Applicants employed a simple computational approach todelineate three new variants of CRISPR-Cas systems but many more arelikely to exist bacterial genomes that have not yet been sequenced.Although most if not all of these new CRISPR-Cas systems are expected tobe rare, they could employ novel strategies and molecular mechanisms andwould provide a major resource for new applications in genomeengineering and biotechnology.

TBLASTN program with the E-value cut-off of 0.01 and low complexityfiltering turned off parameters was used to search with Cas1 profile(Makarova et al., 2015) as a query against NCBI WGS database. Sequencesof contigs or complete genome partitions where Cas1 hit has beenidentified were retrieved from the same database. The region around theCas1 gene (the region 20 kb from the start of the Cas1 gene and 20 kbfrom the end of the Cas1 gene) was extracted and translated usingGeneMarkS (Besemer et al., 2001, supra). Predicted proteins from eachCas1-encoding region were searched against a collection of profiles fromCDD database (Marchler-Bauer, 2009) and specific Cas protein profiles(Makarova et al., 2015) using the RPS-BLAST program (Marchler-Bauer etal., 2002, Nucleic Acids Res, vol. 30, 281-283). Procedure to identifycompleteness of CRISPR loci and to classify CRISPR-Cas systems into theexisting types and subtypes (Makarova et al., 2015) developed previouslyhas been applied to each locus.

CRISPRmap (Lange, 2013) was used for repeat classification.

Partial and/or unclassified loci that encompassed proteins larger than500 amino acids were analyzed on a case-by-case basis. Specifically,each predicted protein encoded in these loci was searched usingiterative profile searches with the PSI-BLAST (Altschul, 1997), with acut-off e-value of 0.01 and composition based-statistics and lowcomplexity filtering turned off, to search for distantly similarsequences against NCBI's non-redundant (NR) protein sequence database.Each identified non-redundant protein was searched against WGS databaseusing the TBLAST program (Altschul, 1997). The HHpred program was usedwith default parameters to identify remote sequence similarity (Soding,2005) using as the queries all proteins identified in the BLASTsearches. Multiple sequence alignments were constructed using MUSCLE(Edgar, 2004) and MAFFT (Katoh and Standley, 2013, Mol Biol Evol, vol.30, 772-780). Phylogenetic analysis was performed using the FastTreeprogram with the WAG evolutionary model and the discrete gamma modelwith 20 rate categories (Price et al., 2010, PLoS One, vol. 5, e9490).Protein secondary structure was predicted using Jpred 4 (Drozdetskiy,2015).

CRISPR repeats were identified using PILER-CR (Edgar, 2007, supra) or,for degenerate repeats, CRISPRfinder (Grissa et al., 2007, Nucleic AcidsRes, vol. 35, W52-57). The Mfold program (Zuker, 2003, Nucleic AcidsRes, vol. 31, 3406-3415) was used to identify the most stable structurefor the repeat sequences.

The spacer sequences were searched against the NCBI nucleotide NR andWGS databases using MEGABLAST (Morgulis et al., 2008, Bioinformatics,vol. 24, 1757-1764) with default parameters except that the word sizewas set at 20.

Chosen Gene Candidates

Gene ID: A; Gene Type: C2C1; Organism: 5. Opitutaceae bacterium TAV5;Spacer Length - mode (range): 34 (33 to 37); DR1: (SEQ ID NO: 38)GCCGCAGCGAAUGCCGUUUCACGAAUCGUCAGGCGG; DR2: none; tracrRNA1:(SEQ ID NO: 39) GCUGGAGACGUUUUUUGAAACGGCGAGUGCUGCGGAUAGCGAGUUUCUCUUGGGGAGGCGCUCGCGGCCACUUUU; tracrRNA2: none; Protein Sequence:(SEQ ID NO: 40) MSLNRIYQGRVAAVETGTALAKGNVEWMPAAGGDEVLWQHHELFQAAINYYLVALLALADKNNPVLGPLISQMDNPQSPYHVWGSFRRQGRQRTGLSQAVAPYITPGNNAPTLDEVFRSILAGNPTDRATLDAALMQLLKACDGAGAIQQEGRSYWPKFCDPDSTANFAGDPAMLRREQHRLLLPQVLHDPAITHDSPALGSFDTYSIATPDTRTPQLTGPKARARLEQAITLWRVRLPESAADFDRLASSLKKIPDDDSRLNLQGYVGSSAKGEVQARLFALLLFRHLERSSFTLGLLRSATPPPKNAETPPPAGVPLPAASAADPVRIARGKRSFVFRAFTSLPCWHGGDNIHPTWKSFDIAAFKYALTVINQIEEKTKERQKECAELETDFDYMHGRLAKIPVKYTTGEAEPPPILANDLRIPLLRELLQNIKVDTALTDGEAVSYGLQRRTIRGFRELRRIWRGHAPAGTVFSSELKEKLAGELRQFQTDNSTTIGSVQLFNELIQNPKYWPIWQAPDVETARQWADAGFADDPLAALVQEAELQEDIDALKAPVKLTPADPEYSRRQYDFNAVSKFGAGSRSANRHEPGQTERGHNTFTTEIAARNAADGNRWRATHVRIHYSAPRLLRDGLRRPDTDGNEALEAVPWLQPMMEALAPLPTLPQDLTGMPVFLMPDVTLSGERRILLNLPVTLEPAALVEQLGNAGRWQNQFFGSREDPFALRWPADGAVKTAKGKTHIPWHQDRDHFTVLGVDLGTRDAGALALLNVTAQKPAKPVHRIIGEADGRTWYASLADARMIRLPGEDARLFVRGKLVQEPYGERGRNASLLEWEDARNIILRLGQNPDELLGADPRRHSYPEINDKLLVALRRAQARLARLQNRSWRLRDLAESDKALDEIHAERAGEKPSPLPPLARDDAIKSTDEALLSQRDIIRRSFVQIANLILPLRGRRWEWRPHVEVPDCHILAQSDPGTDDTKRLVAGQRGISHERIEQIEELRRRCQSLNRALRHKPGERPVLGRPAKGEEIADPCPALLEKINRLRDQRVDQTAHAILAAALGVRLRAPSKDRAERRHRDIHGEYERFRAPADFVVIENLSRYLSSQDRARSENTRLMQWCHRQIVQKLRQLCETYGIPVLAVPAAYSSRFSSRDGSAGFRAVHLTPDHRHRMPWSRILARLKAHEEDGKRLEKTVLDEARAVRGLFDRLDRFNAGHVPGKPWRTLLAPLPGGPVFVPLGDATPMQADLNAAINIALRGIAAPDRHDIHHRLRAENKKRILSLRLGTQREKARWPGGAPAVTLSTPNNGASPEDSDALPERVSNLFVDIAGVANFERVTIEGVSQKFATGRGLWASVKQRAWNRVARLNETVTDNNRNEEEDDIPMGene ID: B; Gene Type: C2C1; Organism:7. Bacillus thermoamylovorans strain B4166; Spacer Length - mode(range): 37 (35-38); DR1: (SEQ ID NO: 41)GUCCAAGAAAAAAGAAAUGAUACGAGGCAUUAGCAC; DR2: none; tracrRNA1:(SEQ ID NO: 42) CUGGACGAUGUCUCUUUUAUUUCUUUUUUCUUGGAUCUGAGUACGAGCACCCACAUUGGACAUUUCGCAUGGUGGGUGCUCGUACUAUAGGUAAAACAAACCUUUUU;tracrRNA2: none; Protein Sequence: (SEQ ID NO: 43)MATRSFILKIEPNEEVKKGLWKTHEVLNHGIAYYMNILKLIRQEAIYEHHEQDPKNPKKVSKAEIQAELWDFVLKMQKCNSFTHEVDKDVVFNILRELYEELVPSSVEKKGEANQLSNKFLYPLVDPNSQSGKGTASSGRKPRWYNLKIAGDPSWEEEKKKWEEDKKKDPLAKILGKLAEYGLIPLFIPFTDSNEPIVKEIKWMEKSRNQSVRRLDKDMFIQALERFLSWESWNLKVKEEYEKVEKEHKTLEERIKEDIQAFKSLEQYEKERQEQLLRDTLNTNEYRLSKRGLRGWREIIQKWLKMDENEPSEKYLEVFKDYQRKHPREAGDYSVYEFLSKKENHFIWRNHPEYPYLYATFCEIDKKKKDAKQQATFTLADPINHPLWVRFEERSGSNLNKYRILTEQLHTEKLKKKLTVQLDRLIYPTESGGWEEKGKVDIVLLPSRQFYNQIFLDIEEKGKHAFTYKDESIKFPLKGTLGGARVQFDRDHLRRYPHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDFPKFVNFKPKELTEWIKDSKGKKLKSGIESLEIGLRVMSIDLGQRQAAAASIFEVVDQKPDIEGKLFFPIKGTELYAVHRASFNIKLPGETLVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQQFEDITEREKRVTKWISRQENSDVPLVYQDELIQIRELMYKPYKDWVAFLKQLHKRLEVEIGKEVKHWRKSLSDGRKGLYGISLKNIDEIDRTRKFLLRWSLRPTEPGEVRRLEPGQRFAIDQLNHLNALKEDRLKKMANTIIMHALGYCYDVRKKKWQAKNPACQIILFEDLSNYNPYEERSRFENSKLMKWSRREIPRQVALQGEIYGLQVGEVGAQFSSRFHAKTGSPGIRCSVVTKEKLQDNRFFKNLQREGRLTLDKIAVLKEGDLYPDKGGEKFISLSKDRKLVTTHADINAAQNLQKRFWTRTHGFYKVYCKAYQVDGQTVYIPESKDQKQKIIEEFGEGYFILKDGVYEWGNAGKLKIKKGSSKQSSSELVDSDILKDSFDLASELKGEKLMLYRDPSGNVFPSDKWMAAGVFFGKLERILISKLTNQYSISTIEDDSSKQSMGene ID: C; Gene Type: C2C1; Organism: 9. Bacillus sp. NSP2.1;Spacer Length - mode (range): 36 (35-42); DR1: (SEQ ID NO: 44)GUUCGAAAGCUUAGUGGAAAGCUUCGUGGUUAGCAC; DR2: none; tracrRNA1:(SEQ ID NO: 45) CACGGAUAAUCACGACUUUCCACUAAGCUUUCGAAUUUUAUGAUGCGAGCAUCCUCUCAGGUCAAAAAA; tracrRNA2: none; Protein Sequence: (SEQ ID NO: 46)MAIRSIKLKLKTHTGPEAQNLRKGIWRTHRLLNEGVAYYMKMLLLFRQESTGERPKEELQEELICHIREQQQRNQADKNTQALPLDKALEALRQLYELLVPSSVGQSGDAQIISRKFLSPLVDPNSEGGKGTSKAGAKPTWQKKKEANDPTWEQDYEKWKKRREEDPTASVITTLEEYGIRPIFPLYTNTVTDIAWLPLQSNQFVRTWDRDMLQQAIERLLSWESWNKRVQEEYAKLKEKMAQLNEQLEGGQEWISLLEQYEENRERELRENMTAANDKYRITKRQMKGWNELYELWSTFPASASHEQYKEALKRVQQRLRGRFGDAHFFQYLMEEKNRLIWKGNPQRIHYFVARNELTKRLEEAKQSATMTLPNARKHPLWVRFDARGGNLQDYYLTAEADKPRSRRFVTFSQLIWPSESGWMEKKDVEVELALSRQFYQQVKLLKNDKGKQKIEFKDKGSGSTFNGHLGGAKLQLERGDLEKEEKNFEDGEIGSVYLNVVIDFEPLQEVKNGRVQAPYGQVLQLIRRPNEFPKVTTYKSEQLVEWIKASPQHSAGVESLASGFRVMSIDLGLRAAAATSIFSVEESSDKNAADFSYWIEGTPLVAVHQRSYMLRLPGEQVEKQVMEKRDERFQLHQRVKFQIRVLAQIMRMANKQYGDRWDELDSLKQAVEQKKSPLDQTDRTFWEGIVCDLTKVLPRNEADWEQAVVQIHRKAEEYVGKAVQAWRKRFAADERKGIAGLSMWNIEELEGLRKLLISWSRRTRNPQEVNRFERGHTSHQRLLTHIQNVKEDRLKQLSHAIVMTALGYVYDERKQEWCAEYPACQVILFENLSQYRSNLDRSTKENSTLMKWAHRSIPKYVHMQAEPYGIQIGDVRAEYSSRFYAKTGTPGIRCKKVRGQDLQGRRFENLQKRLVNEQFLTEEQVKQLRPGDIVPDDSGELFMTLTDGSGSKEVVFLQADINAAHNLQKRFWQRYNELFKVSCRVIVRDEEEYLVPKTKSVQAKLGKGLFVKKSDTAWKDVYVWDSQAKLKGKTTFTEESESPEQLEDFQEIIEEAEEAKGTYRTLFRDPSGVFFPESVWYPQKDFWGEVKRKLYGKLRERFLTKARGene ID: D; Gene Type: C2C2; Organism:4. Lachnospiraceae bacterium NK4A144 G619; Spacer Length -mode (range): 35; DR1: (SEQ ID NO: 47)GUUUUGAGAAUAGCCCGACAUAGAGGGCAAUAGAC; DR2: (SEQ ID NO: 48)GUUAUGAAAACAGCCCGACAUAGAGGGCAAUAGACA; (SEQ ID NO: 49)tracrRNA1: none; tracrRNA2: none; Protein Sequence:MKISKVDHTRMAVAKGNQHRRDEISGILYKDPTKTGSIDFDERFKKLNCSAKILYHVFNGIAEGSNKYKNIVDKVNNNLDRVLFTGKSYDRKSIIDIDTVLRNVEKINAFDRISTEEREQIIDDLLEIQLRKGLRKGKAGLREVLLIGAGVIVRTDKKQEIADFLEILDEDFNKTNQAKNIKLSIENQGLVVSPVSRGEERIFDVSGAQKGKSSKKAQEKEALSAFLLDYADLDKNVRFEYLRKIRRLINLYFYVKNDDVMSLTEIPAEVNLEKDFDIWRDHEQRKEENGDFVGCPDILLADRDVKKSNSKQVKIAERQLRESIREKNIKRYRFSIKTIEKDDGTYFFANKQISVFWIHRIENAVERILGSINDKKLYRLRLGYLGEKVWKDILNFLSIKYIAVGKAVFNFAMDDLQEKDRDIEPGKISENAVNGLTSFDYEQIKADEMLQREVAVNVAFAANNLARVTVDIPQNGEKEDILLWNKSDIKKYKKNSKKGILKSILQFFGGASTWNMKMFEIAYHDQPGDYEENYLYDIIQIIYSLRNKSFHFKTYDHGDKNWNRELIGKMIEHDAERVISVEREKFHSNNLPMFYKDADLKKILDLLYSDYAGRASQVPAFNTVLVRKNFPEFLRKDMGYKVHFNNPEVENQWHSAVYYLYKEIYYNLFLRDKEVKNLFYTSLKNIRSEVSDKKQKLASDDFASRCEEIEDRSLPEICQIIMTEYNAQNFGNRKVKSQRVIEKNKDIFRHYKMLLIKTLAGAFSLYLKQERFAFIGKATPIPYETTDVKNFLPEWKSGMYASFVEEIKNNLDLQEWYIVGRFLNGRMLNQLAGSLRSYIQYAEDIERRAAENRNKLFSKPDEKIEACKKAVRVLDLCIKISTRISAEFTDYFDSEDDYADYLEKYLKYQDDAIKELSGSSYAALDHFCNKDDLKFDIYVNAGQKPILQRNIVMAKLFGPDNILSEVMEKVTESAIREYYDYLKKVSGYRVRGKCSTEKEQEDLLKFQRLKNAVEFRDVTEYAEVINELLGQLISWSYLRERDLLYFQLGFHYMCLKNKSFKPAEYVDIRRNNGTIIHNAILYQIVSMYINGLDFYSCDKEGKTLKPIETGKGVGSKIGQFIKYSQYLYNDPSYKLEIYNAGLEVFENIDEHDNITDLRKYVDHFKYYAYGNKMSLLDLYSEFFDRFFTYDMKYQKNVVNVLENILLRHFVIFYPKFGSGKKDVGIRDCKKERAQIEISEQSLTSEDFNIFKLDDKAGEEAKKFPARDERYLQTIAKLLYYPNEIEDMNRFMKKGETINKKVQFNRKKKITRKQKNNSSNEVLSSTMGYLFKNIKL Gene ID: E; Gene Type: C2C2; Organism:8. Listeria seeligeri serovar 1/2b str. SLCC3954; Spacer Length -mode (range): 30; DR1: (SEQ ID NO: 50)GUUUUAGUCCUCUUUCAUAUAGAGGUAGUCUCUUAC; DR2: none; tracrRNA1 :(SEQ ID NO: 51) AUGAAAAGAGGACUAAAACUGAAAGAGGACUAAAACACCAGAUGUGGAUAACUAUAUUAGUGGCUAUUAAAAAUUCGUCGAUAUUAGAGAGGAAACUUU;tracrRNA2: none; Protein Sequence: (SEQ ID NO: 52)MWISIKTLIHHLGVLFFCDYMYNRREKKIIEVKTMRITKVEVDRKKVLISRDKNGGKLVYENEMQDNTEQIMHEIKKSSFYKSVVNKTICRPEQKQMKKLVHGLLQENSQEKIKVSDVTKLNISNFLNHRFKKSLYYFPENSPDKSEEYRIEINLSQLLEDSLKKQQGTFICWESFSKDMELYINWAENYISSKTKLIKKSIRNNRIQSTESRSGQLMDRYMKDILNKNKPFDIQSVSEKYQLEKLTSALKATFKEAKKNDKEINYKLKSTLQNHERQIIEELKENSELNQFNIEIRKHLETYFPIKKTNRKVGDIRNLEIGEIQKIVNHRLKNKIVQRILQEGKLASYEIESTVNSNSLQKIKIEEAFALKFINACLFASNNLRNMVYPVCKKDILMIGEFKNSFKEIKHKKFIRQWSQFFSQEITVDDIELASWGLRGAIAPIRNEIIHLKKHSWKKFFNNPTFKVKKSKIINGKTKDVTSEFLYKETLFKDYFYSELDSVPELIINKMESSKILDYYSSDQLNQVFTIPNFELSLLTSAVPFAPSFKRVYLKGFDYQNQDEAQPDYNLKLNIYNEKAFNSEAFQAQYSLFKMVYYQVFLPQFTTNNDLFKSSVDFILTLNKERKGYAKAFQDIRKMNKDEKPSEYMSYIQSQLMLYQKKQEEKEKINHFEKFINQVFIKGFNSFIEKNRLTYICHPTKNTVPENDNIEIPFHTDMDDSNIAFWLMCKLLDAKQLSELRNEMIKFSCSLQSTEEISTFTKAREVIGLALLNGEKGCNDWKELFDDKEAWKKNMSLYVSEELLQSLPYTQEDGQTPVINRSIDLVKKYGTETILEKLFSSSDDYKVSAKDIAKLHEYDVTEKIAQQESLHKQWIEKPGLARDSAWTKKYQNVINDISNYQWAKTKVELTQVRHLHQLTIDLLSRLAGYMSIADRDFQFSSNYILERENSEYRVTSWILLSENKNKNKYNDYELYNLKNASIKVSSKNDPQLKVDLKQLRLTLEYLELFDNRLKEKRNNISHFNYLNGQLGNSILELFDDARDVLSYDRKLKNAVSKSLKEILSSHGMEVTFKPLYQTNHHLKIDKLQPKKIHHLGEKSTVSSNQVSNEYCQLVRTLLTMKGene ID: F; Gene Type: C2C2; Organism: 12. Leptotrichia wadei F0279;Spacer Length - mode (range): 31; DR1: (SEQ ID NO: 53)GUUUUAGUCCCCUUCGUUUUUGGGGUAGUCUAAAUC; DR2: none; tracrRNA1:(SEQ ID NO: 54) GAUUUAGAGCACCCCAAAAGUAAUGAAAAUUUGCAAUUAAAUAAGGAAUAUUAAAAAAAUGUGAUUUUAAAAAAAUUGAAGAAAUUAAAUGAAAAAUUGUCCAAGUAA AAAAA; tracrRNA2:(SEQ ID NO: 55) AUUUAGAUUACCCCUUUAAUUUAUUUUACCAUAUUUUUCUCAUAAUGCAAACUAAUAUUCCAAAAUUUUU; Protein Sequence: (SEQ ID NO: 56)MGNLFGHKRWYEVRDKKDFKIKRKVKVKRNYDGNKYILNINENNNKEKIDNNKFIRKYINYKKNDNILKEFTRKFHAGNILFKLKGKEGIIRIENNDDFLETEEVVLYIEAYGKSEKLKALGITKKKIIDEAIRQGITKDDKKIEIKRQENEEEIEIDIRDEYTNKTLNDCSIILRIIENDELETKKSIYEIFKNINMSLYKIIEKIIENETEKVFENRYYEEHLREKLLKDDKIDVILTNFMEIREKIKSNLEILGFVKFYLNVGGDKKKSKNKKMLVEKILNINVDLTVEDIADFVIKELEFWNITKRIEKVKKVNNEFLEKRRNRTYIKSYVLLDKHEKFKIERENKKDKIVKFFVENIKNNSIKEKIEKILAEFKIDELIKKLEKELKKGNCDTEIFGIFKKHYKVNFDSKKFSKKSDEEKELYKIIYRYLKGRIEKILVNEQKVRLKKMEKIEIEKILNESILSEKILKRVKQYTLEHIMYLGKLRHNDIDMTTVNTDDFSRLHAKEELDLELITFFASTNMELNKIFSRENINNDENIDFFGGDREKNYVLDKKILNSKIKIIRDLDFIDNKNNITNNFIRKFTKIGTNERNRILHAISKERDLQGTQDDYNKVINIIQNLKISDEEVSKALNLDVVFKDKKNIITKINDIKISEENNNDIKYLPSFSKVLPEILNLYRNNPKNEPFDTIETEKIVLNALIYVNKELYKKLILEDDLEENESKNIFLQELKKTLGNIDEIDENIIENYYKNAQISASKGNNKAIKKYQKKVIECYIGYLRKNYEELFDFSDFKMNIQEIKKQIKDINDNKTYERITVKTSDKTIVINDDFEYIISIFALLNSNAVINKIRNRFFATSVWLNTSEYQNIIDILDEIMQLNTLRNECITENWNLNLEEFIQKMKEIEKDFDDFKIQTKKEIFNNYYEDIKNNILTEFKDDINGCDVLEKKLEKIVIFDDETKFEIDKKSNILQDEQRKLSNINKKDLKKKVDQYIKDKDQEIKSKILCRIIFNSDFLKKYKKEIDNLIEDMESENENKFQEIYYPKERKNELYIYKKNLFLNIGNPNFDKIYGLISNDIKMADAKFLFNIDGKNIRKNKISEIDAILKNLNDKLNGYSKEYKEKYIKKLKENDDFFAKNIQNKNYKSFEKDYNRVSEYKKIRDLVEFNYLNKIESYLIDINWKLAIQMARFERDMHYIVNGLRELGIIKLSGYNTGISRAYPKRNGSDGFYTTTAYYKFFDEESYKKFEKICYGFGIDLSENSEINKPENESIRNYISHFYIVRNPFADYSIAEQIDRVSNLLSYSTRYNNSTYASVFEVFKKDVNLDYDELKKKFKLIGNNDILERLMKPKKVSVLELESYNSDYIKNLIIELLTKIENTNDTLGene ID: G; Gene Type: C2C2; Organism: 14. Leptotrichia shahii DSM19757 B031; Spacer Length - mode (range): 30 (30-32); DR1:(SEQ ID NO: 57) GUUUUAGUCCCCUUCGAUAUUGGGGUGGUCUAUAUC;DR2: none; tracrRNA1: (SEQ ID NO: 58)AUUGAUGUGGUAUACUAAAAAUGGAAAAUUGUAUUUUUGAUUAGAAAGAUGUAAAAUUGAUUUAAUUUAAAAAUAUUUUAUUAGAUUAAAGUAGA;tracrRNA2: none; Protein Sequence: (SEQ ID NO: 59)MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNNGene ID: H; Gene Type: Cp1; Organism: Francisella ularensis subsp.novicida U112; Spacer Length - mode (range): 31; DR1: (SEQ ID NO: 60)GUCUAAGAACUUUAAAUAAUUUCUACUGUUGUAGAU; DR2: none; tracrRNA1:(SEQ ID NO: 61) AUCUACAAAAUUAUAAACUAAAUAAAGAUUCUUAUAAUAACUUUAUAUAUAAUCGAAAUGUAGAGAAUUUU; tracrRNA2: none; Protein Sequence: (SEQ ID NO: 62)MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKEFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN

Genes for Synthesis

For genes A through H, the Applicants optimize the genes for humanexpression and append the following DNA sequence to the end of eachgene. Note this DNA sequence contains a stop codon (underlined), so nostop codon is added to the codon optimized gene sequence:

(SEQ ID NO: 63) AAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGggatccTACCCATACGATGTTCCAGATTACGCTTATCCCTACGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCCTAA

For optimization, avoid the following restriction sites: BamHI, EcoRI,HindIII, BsmBI, BsaI, BbsI, AgeI, XhoI, NdeI, NotI, KpnI, BsrGI, SpeI,XbaI, NheI

These genes are cloned into a simple mammalian expression vector:

>A (SEQ ID NO: 64) MSLNRIYQGRVAAVETGTALAKGNVEWMPAAGGDEVLWQHHELFQAAINYYLVALLALADKNNPVLGPLISQMDNPQSPYHVWGSFRRQGRQRTGLSQAVAPYITPGNNAPTLDEVFRSILAGNPTDRATLDAALMQLLKACDGAGAIQQEGRSYWPKFCDPDSTANEAGDPAMLRREQHRLLLPQVLHDPAITHDSPALGSFDTYSIATPDTRTPQLTGPKARARLEQAITLWRVRLPESAADFDRLASSLKKIPDDDSRLNLQGYVGSSAKGEVQARLFALLLFRHLERSSFTLGLLRSATPPPKNAETPPPAGVPLPAASAADPVRIARGKRSFVFRAFTSLPCWHGGDNIHPTWKSFDIAAFKYALTVINQIEEKTKERQKECAELETDFDYMHGRLAKIPVKYTTGEAEPPPILANDLRIPLLRELLQNIKVDTALTDGEAVSYGLQRRTIRGFRELRRIWRGHAPAGTVFSSELKEKLAGELRQFQTDNSTTIGSVQLFNELIQNPKYWPIWQAPDVETARQWADAGFADDPLAALVQEAELQEDIDALKAPVKLTPADPEYSRRQYDFNAVSKFGAGSRSANRHEPGQTERGHNTFTTEIAARNAADGNRWRATHVRIHYSAPRLLRDGLRRPDTDGNEALEAVPWLQPMMEALAPLPTLPQDLTGMPVFLMPDVTLSGERRILLNLPVTLEPAALVEQLGNAGRWQNQFFGSREDPFALRWPADGAVKTAKGKTHIPWHQDRDHFTVLGVDLGTRDAGALALLNVTAQKPAKPVHRIIGEADGRTWYASLADARMIRLPGEDARLFVRGKLVQEPYGERGRNASLLEWEDARNIILRLGQNPDELLGADPRRHSYPEINDKLLVALRRAQARLARLQNRSWRLRDLAESDKALDEIHAERAGEKPSPLPPLARDDAIKSTDEALLSQRDIIRRSFVQIANLILPLRGRRWEWRPHVEVPDCHILAQSDPGTDDTKRLVAGQRGISHERIEQIEELRRRCQSLNRALRHKPGERPVLGRPAKGEEIADPCPALLEKINRLRDQRVDQTAHAILAAALGVRLRAPSKDRAERRHRDIHGEYERFRAPADFVVIENLSRYLSSQDRARSENTRLMQWCHRQIVQKLRQLCETYGIPVLAVPAAYSSRFSSRDGSAGFRAVHLTPDHRHRMPWSRILARLKAHEEDGKRLEKTVLDEARAVRGLFDRLDRFNAGHVPGKPWRTLLAPLPGGPVFVPLGDATPMQADLNAAINIALRGIAAPDRHDIHHRLRAENKKRILSLRLGTQREKARWPGGAPAVTLSTPNNGASPEDSDALPERVSNLFVDIAGVANFERVTIEGVSQKFATGRGLWASVKQRAWNRVARLNETVTDNNRNEEEDDIPM >B (SEQ ID NO: 65)MATRSFILKIEPNEEVKKGLWKTHEVLNHGIAYYMNILKLIRQEAIYEHHEQDPKNPKKVSKAEIQAELWDFVLKMQKCNSFTHEVDKDVVFNILRELYEELVPSSVEKKGEANQLSNKFLYPLVDPNSQSGKGTASSGRKPRWYNLKIAGDPSWEEEKKKWEEDKKKDPLAKILGKLAEYGLIPLFIPFTDSNEPIVKEIKWMEKSRNQSVRRLDKDMFIQALERFLSWESWNLKVKEEYEKVEKEHKTLEERIKEDIQAFKSLEQYEKERQEQLLRDTLNTNEYRLSKRGLRGWREIIQKWLKMDENEPSEKYLEVFKDYQRKHPREAGDYSVYEFLSKKENHFIWRNHPEYPYLYATFCEIDKKKKDAKQQATFTLADPINHPLWVRFEERSGSNLNKYRILTEQLHTEKLKKKLTVQLDRLIYPTESGGWEEKGKVDIVLLPSRQFYNQIFLDIEEKGKHAFTYKDESIKFPLKGTLGGARVQFDRDHLRRYPHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDEPKFVNFKPKELTEWIKDSKGKKLKSGIESLEIGLRVMSIDLGQRQAAAASIFEVVDQKPDIEGKLFFPIKGTELYAVHRASFNIKLPGETLVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQQFEDITEREKRVTKWISRQENSDVPLVYQDELIQIRELMYKPYKDWVAFLKQLHKRLEVEIGKEVKHWRKSLSDGRKGLYGISLKNIDEIDRTRKFLLRWSLRPTEPGEVRRLEPGQRFAIDQLNHLNALKEDRLKKMANTIIMHALGYCYDVRKKKWQAKNPACQIILFEDLSNYNPYEERSRFENSKLMKWSRREIPRQVALQGEIYGLQVGEVGAQFSSRFHAKTGSPGIRCSVVTKEKLQDNRFFKNLQREGRLTLDKIAVLKEGDLYPDKGGEKFISLSKDRKLVTTHADINAAQNLQKRFWTRTHGFYKVYCKAYQVDGQTVYIPESKDQKQKIIEEFGEGYFILKDGVYEWGNAGKLKIKKGSSKQSSSELVDSDILKDSFDLASELKGEKLMLYRDPSGNVFPSDKWMAAGVFFGKLERILISKLTNQYSISTIEDDSSKQSM >C (SEQ ID NO: 66)MAIRSIKLKLKTHTGPEAQNLRKGIWRTHRLLNEGVAYYMKMLLLFRQESTGERPKEELQEELICHIREQQQRNQADKNTQALPLDKALEALRQLYELLVPSSVGQSGDAQIISRKFLSPLVDPNSEGGKGTSKAGAKPTWQKKKEANDPTWEQDYEKWKKRREEDPTASVITTLEEYGIRPIFPLYTNTVTDIAWLPLQSNQFVRTWDRDMLQQAIERLLSWESWNKRVQEEYAKLKEKMAQLNEQLEGGQEWISLLEQYEENRERELRENMTAANDKYRITKRQMKGWNELYELWSTFPASASHEQYKEALKRVQQRLRGRFGDAHFFQYLMEEKNRLIWKGNPQRIHYFVARNELTKRLEEAKQSATMTLPNARKHPLWVRFDARGGNLQDYYLTAEADKPRSRRFVTFSQLIWPSESGWMEKKDVEVELALSRQFYQQVKLLKNDKGKQKIEFKDKGSGSTFNGHLGGAKLQLERGDLEKEEKNFEDGEIGSVYLNVVIDFEPLQEVKNGRVQAPYGQVLQLIRRPNEFPKVTTYKSEQLVEWIKASPQHSAGVESLASGFRVMSIDLGLRAAAATSIFSVEESSDKNAADFSYWIEGTPLVAVHQRSYMLRLPGEQVEKQVMEKRDERFQLHQRVKFQIRVLAQIMRMANKQYGDRWDELDSLKQAVEQKKSPLDQTDRTFWEGIVCDLTKVLPRNEADWEQAVVQIHRKAEEYVGKAVQAWRKRFAADERKGIAGLSMWNIEELEGLRKLLISWSRRTRNPQEVNRFERGHTSHQRLLTHIQNVKEDRLKQLSHAIVMTALGYVYDERKQEWCAEYPACQVILFENLSQYRSNLDRSTKENSTLMKWAHRSIPKYVHMQAEPYGIQIGDVRAEYSSRFYAKTGTPGIRCKKVRGQDLQGRRFENLQKRLVNEQFLTEEQVKQLRPGDIVPDDSGELFMTLTDGSGSKEVVFLQADINAAHNLQKRFWQRYNELFKVSCRVIVRDEEEYLVPKTKSVQAKLGKGLFVKKSDTAWKDVYVWDSQAKLKGKTTFTEESESPEQLEDFQEIIEEAEEAKGTYRTLFRDPSGVFFPESVWYPQKDFWGEVKRKLYGKLRERF LTKAR >D(SEQ ID NO: 67) MKISKVDHTRMAVAKGNQHRRDEISGILYKDPTKTGSIDFDERFKKLNCSAKILYHVFNGIAEGSNKYKNIVDKVNNNLDRVLFTGKSYDRKSIIDIDTVLRNVEKINAFDRISTEEREQIIDDLLEIQLRKGLRKGKAGLREVLLIGAGVIVRTDKKQEIADFLEILDEDFNKTNQAKNIKLSIENQGLVVSPVSRGEERIFDVSGAQKGKSSKKAQEKEALSAFLLDYADLDKNVRFEYLRKIRRLINLYFYVKNDDVMSLTEIPAEVNLEKDFDIWRDHEQRKEENGDFVGCPDILLADRDVKKSNSKQVKIAERQLRESIREKNIKRYRFSIKTIEKDDGTYFFANKQISVFWIHRIENAVERILGSINDKKLYRLRLGYLGEKVWKDILNFLSIKYIAVGKAVFNFAMDDLQEKDRDIEPGKISENAVNGLTSFDYEQIKADEMLQREVAVNVAFAANNLARVTVDIPQNGEKEDILLWNKSDIKKYKKNSKKGILKSILQFFGGASTWNMKMFEIAYHDQPGDYEENYLYDIIQIIYSLRNKSFHFKTYDHGDKNWNRELIGKMIEHDAERVISVEREKFHSNNLPMFYKDADLKKILDLLYSDYAGRASQVPAFNTVLVRKNFPEFLRKDMGYKVHFNNPEVENQWHSAVYYLYKEIYYNLFLRDKEVKNLFYTSLKNIRSEVSDKKQKLASDDFASRCEEIEDRSLPEICQIIMTEYNAQNFGNRKVKSQRVIEKNKDIFRHYKMLLIKTLAGAFSLYLKQERFAFIGKATPIPYETTDVKNFLPEWKSGMYASFVEEIKNNLDLQEWYIVGRFLNGRMLNQLAGSLRSYIQYAEDIERRAAENRNKLFSKPDEKIEACKKAVRVLDLCIKISTRISAEFTDYFDSEDDYADYLEKYLKYQDDAIKELSGSSYAALDHFCNKDDLKFDIYVNAGQKPILQRNIVMAKLFGPDNILSEVMEKVTESAIREYYDYLKKVSGYRVRGKCSTEKEQEDLLKFQRLKNAVEFRDVTEYAEVINELLGQLISWSYLRERDLLYFQLGFHYMCLKNKSFKPAEYVDIRRNNGTIIHNAILYQIVSMYINGLDFYSCDKEGKTLKPIETGKGVGSKIGQFIKYSQYLYNDPSYKLEIYNAGLEVFENIDEHDNITDLRKYVDHFKYYAYGNKMSLLDLYSEFFDRFFTYDMKYQKNVVNVLENILLRHFVIFYPKFGSGKKDVGIRDCKKERAQIEISEQSLTSEDFMFKLDDKAGEEAKKFPARDERYLQTIAKLLYYPNEIEDMNRFMKKGETINKKVQFNRKKKITRKQKNNSSNEVLSSTMGYLFKNIKL >E (SEQ ID NO: 68)MWISIKTLIHHLGVLFFCDYMYNRREKKIIEVKTMRITKVEVDRKKVLISRDKNGGKLVYENEMQDNTEQIMHHKKSSFYKSVVNKTICRPEQKQMKKLVHGLLQENSQEKIKVSDVTKLNISNFLNHRFKKSLYYFPENSPDKSEEYRIEINLSQLLEDSLKKQQGTFICWESFSKDMELYINWAENYISSKTKLIKKSIRNNRIQSTESRSGQLMDRYMKDILNKNKPFDIQSVSEKYQLEKLTSALKATFKEAKKNDKEINYKLKSTLQNHERQIIEELKENSELNQFNIEIRKHLETYFPIKKTNRKVGDIRNLEIGEIQKIVNHRLKNKIVQRILQEGKLASYEIESTVNSNSLQKIKIEEAFALKFINACLFASNNLRNMVYPVCKKDILMIGEFKNSEKEIKHKKFIRQWSQFFSQEITVDDIELASWGLRGAIAPIRNEIIHLKKHSWKKFFNNPTFKVKKSKIINGKTKDVTSEFLYKETLFKDYFYSELDSVPELIINKMESSKILDYYSSDQLNQVFTIPNFELSLLTSAVPFAPSFKRVYLKGFDYQNQDEAQPDYNLKLNIYNEKAFNSEAFQAQYSLFKMVYYQVFLPQFTTNNDLFKSSVDFILTLNKERKGYAKAFQDIRKMNKDEKPSEYMSYIQSQLMLYQKKQEEKEKINHFEKFINQVFIKGENSFIEKNRLTYICHPTKNTVPENDNIEIPFHTDMDDSNIAFWLMCKLLDAKQLSELRNEMIKFSCSLQSTEEISTFTKAREVIGLALLNGEKGCNDWKELFDDKEAWKKNMSLYVSEELLQSLPYTQEDGQTPVINRSIDLVKKYGTETILEKLFSSSDDYKVSAKDIAKLHEYDVTEKIAQQESLHKQWIEKPGLARDSAWTKKYQNVINDISNYQWAKTKVELTQVRHLHQLTIDLLSRLAGYMSIADRDFQFSSNYILERENSEYRVTSWILLSENKNKNKYNDYELYNLKNASIKVSSKNDPQLKVDLKQLRLTLEYLELFDNRLKEKRNNISHFNYLNGQLGNSILELFDDARDVLSYDRKLKNAVSKSLKEILSSHGMEVTFKPLYQTNHHLKIDKLQPKKIHHLGEKSTVSSNQVSNEYCQLVRTLLTMK >F (SEQ ID NO: 69)MKVTKVDGISHKKYIEEGKLVKSTSEENRTSERLSELLSIRLDIYIKNPDNASEEENRIRRENLKKFFSNKVLHLKDSVLYLKNRKEKNAVQDKNYSEEDISEYDLKNKNSFSVLKKILLNEDVNSEELEIFRKDVEAKLNKINSLKYSFEENKANYQKINENNVEKVGGKSKRNIIYDYYRESAKRNDYINNVQEAFDKLYKKEDIEKLFFLIENSKKHEKYKIREYYHKIIGRKNDKENFAKIIYEEIQNVNNIKELIEKIPDMSELKKSQVFYKYYLDKEELNDKNIKYAFCHFVEIEMSQLLKNYVYKRLSNISNDKIKRIFEYQNLKKLIENKLLNKLDTYVRNCGKYNYYLQVGEIATSDFIARNRQNEAFLRNIIGVSSVAYFSLRNILETENENDITGRMRGKTVKNNKGEEKYVSGEVDKIYNENKQNEVKENLKMFYSYDFNMDNKNEIEDFFANIDEAISSIRHGIVHFNLELEGKDIFAFKNIAPSEISKKMFQNEINEKKLKLKIFKQLNSANVFNYYEKDVIIKYLKNTKFNFVNKNIPFVPSFTKLYNKIEDLRNTLKFFWSVPKDKEEKDAQIYLLKNIYYGEFLNKFVKNSKVFFKITNEVIKINKQRNQKTGHYKYQKFENIEKTVPVEYLAIIQSREMINNQDKEEKNTYIDFIQQIFLKGFIDYLNKNNLKYIESNNNNDNNDIFSKIKIKKDNKEKYDKILKNYEKHNRNKEIPHEINEFVREIKLGKILKYTENLNMFYLILKLLNHKELTNLKGSLEKYQSANKEETFSDELELINLLNLDNNRVTEDFELEANEIGKFLDFNENKIKDRKELKKFDTNKIYFDGENIIKHRAFYNIKKYGMLNLLEKIADKAKYKISLKELKEYSNKKNEIEKNYTMQQNLHRKYARPKKDEKFNDEDYKEYEKAIGNIQKYTHLKNKVEFNELNLLQGLLLKILHRLVGYTSIWERDLRFRLKGEFPENHYIEEIFNFDNSKNVKYKSGQIVEKYINFYKELYKDNVEKRSIYSDKKVKKLKQEKKDLYIRNYIAHFNYIPHAEISLLEVLENLRKLLSYDRKLKNAIMKSIVDILKEYGFVATFKIGADKKIEIQTLESEKIVHLKNLKKKKLMTDRNSEELCELVKVMFEYKALE >G (SEQ ID NO: 70)MGNLFGHKRWYEVRDKKDFKIKRKVKVKRNYDGNKYILNINENNNKEKIDNNKFIRKYINYKKNDNILKEFTRKFHAGNILFKLKGKEGIIRIENNDDFLETEEVVLYIEAYGKSEKLKALGITKKKIIDEAIRQGITKDDKKIEIKRQENEEEIEIDIRDEYTNKTLNDCSIILRIIENDELETKKSIYEIFKNINMSLYKIIEKIIENETEKVFENRYYEEHLREKLLKDDKIDVILTNFMEIREKIKSNLEILGFVKFYLNVGGDKKKSKNKKMLVEKILNINVDLTVEDIADFVIKELEFWNITKRIEKVKKVNNEFLEKRRNRTYIKSYVLLDKHEKFKIERENKKDKIVKFFVENIKNNSIKEKIEKILAEFKIDELIKKLEKELKKGNCDTEIFGIFKKHYKVNFDSKKFSKKSDEEKELYKIIYRYLKGRIEKILVNEQKVRLKKMEKIEIEKILNESILSEKILKRVKQYTLEHIMYLGKLRHNDIDMTTVNTDDFSRLHAKEELDLELITFFASTNMELNKIFSRENINNDENIDFFGGDREKNYVLDKKILNSKIKIIRDLDFIDNKNNITNNFIRKFTKIGTNERNRILHAISKERDLQGTQDDYNKVINIIQNLKISDEEVSKALNLDVVFKDKKNIITKINDIKISEENNNDIKYLPSFSKVLPEILNLYRNNPKNEPFDTIETEKIVLNALIYVNKELYKKLILEDDLEENESKNIFLQELKKTLGNIDEIDENIIENYYKNAQISASKGNNKAIKKYQKKVIECYIGYLRKNYEELFDFSDFKMNIQEIKKQIKDINDNKTYERITVKTSDKTIVINDDFEYIISIFALLNSNAVINKIRNRFFATSVWLNTSEYQNIIDILDEIMQLNTLRNECITENWNLNLEEFIQKMKEIEKDFDDFKIQTKKEIFNNYYEDIKNNILTEFKDDINGCDVLEKKLEKIVIFDDETKFEIDKKSNILQDEQRKLSNINKKDLKKKVDQYIKDKDQEIKSKILCRIIFNSDFLKKYKKEIDNLIEDMESENENKFQEIYYPKERKNELYIYKKNLFLNIGNPNFDKIYGLISNDIKMADAKFLFNIDGKNIRKNKISEIDAILKNLNDKLNGYSKEYKEKYIKKLKENDDFFAKNIQNKNYKSFEKDYNRVSEYKKIRDLVEFNYLNKIESYLIDINWKLAIQMARFERDMHYIVNGLRELGIIKLSGYNTGISRAYPKRNGSDGFYTTTAYYKFFDEESYKKFEKICYGFGIDLSENSEINKPENESIRNYISHFYIVRNPFADYSIAEQIDRVSNLLSYSTRYNNSTYASVFEVFKKDVNLDYDELKKKFKLIGNNDILERLMKPKKVSVLELESYNSDYIKNLIIELLTKIENTNDTL >H (SEQ ID NO: 71)MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVNINKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN

A-locus through G-locus are cloned and inserted into a low-copy plasmid.A vector that does not contain Amp resistance is used.

>A-locus (SEQ ID NO: 72)TATCCGGTCGAATCGAGAATGACGACCGCTACGTCTTGGACTACGAAGCCGTGGCCCTTGCCGATGCTCTCGGTGTGGATGTTGCCGACCTGTTCCGCAAGATCGATTGCCCCAAGAACCTGCTGCGCAGGCGGGCAGGGTAGGGGAGCGGTTTCCGGCGGAGATTTTCGGAGGCGCCGGTAACGTTATGTCGGGGAATTTGCTATACATCGACGATAATTAGTTTTGTTGATTCAGGATCGAAATGCGCTCAAACAAAGAACGTTCCGCGTTTCCCTCATGCGCTACTACGCCCACACCGCCATCTTTCGGCACGCAAACAAAGCAGATGGGTTGCCTGTCAATGGGTGATCATTGCCTGAAGTTACCATCCATCAATAATATAAATCATCCTTACTCCGAATGTCCCTCAATCGCATCTATCAAGGCCGCGTGGCGGCCGTCGAAACAGGAACGGCCTTAGCGAAAGGTAATGTCGAATGGATGCCTGCCGCAGGAGGCGACGAAGTTCTCTGGCAGCACCACGAACTTTTCCAAGCTGCCATCAACTACTATCTCGTCGCCCTGCTCGCACTCGCCGACAAAAACAATCCCGTACTTGGCCCGCTGATCAGCCAGATGGATAATCCCCAAAGCCCTTACCATGTCTGGGGAAGTTTCCGCCGCCAAGGACGTCAGCGCACAGGTCTCAGTCAAGCCGTTGCACCTTATATCACGCCGGGCAATAACGCTCCCACCCTTGACGAAGTTTTCCGCTCCATTCTTGCGGGCAACCCAACCGACCGCGCAACTTTGGACGCTGCACTCATGCAATTGCTCAAGGCTTGTGACGGCGCGGGCGCTATCCAGCAGGAAGGTCGTTCCTACTGGCCCAAATTCTGCGATCCTGACTCCACTGCCAACTTCGCGGGAGATCCGGCCATGCTCCGGCGTGAACAACACCGCCTCCTCCTTCCGCAAGTTCTCCACGATCCGGCGATTACTCACGACAGTCCTGCCCTTGGCTCGTTCGACACTTATTCGATTGCTACCCCCGACACCAGAACTCCTCAACTCACCGGCCCCAAGGCACGCGCCCGTCTTGAGCAGGCGATCACCCTCTGGCGCGTCCGTCTTCCCGAATCGGCTGCTGACTTCGATCGCCTTGCCAGTTCCCTCAAAAAAATTCCGGACGACGATTCTCGCCTTAACCTTCAGGGCTACGTCGGCAGCAGTGCGAAAGGCGAAGTTCAGGCCCGTCTTTTCGCCCTTCTGCTATTCCGTCACCTGGAGCGTTCCTCCTTTACGCTTGGCCTTCTCCGTTCCGCCACCCCGCCGCCCAAGAACGCTGAAACACCTCCTCCCGCCGGCGTTCCTTTACCTGCGGCGTCCGCAGCCGATCCGGTGCGGATAGCCCGTGGCAAACGCAGTTTTGTTTTTCGCGCATTCACCAGTCTCCCCTGCTGGCATGGCGGTGATAACATCCATCCCACCTGGAAGTCATTCGACATCGCAGCGTTCAAATATGCCCTCACGGTCATCAACCAGATCGAGGAAAAGACGAAAGAACGCCAAAAAGAATGTGCGGAACTTGAAACTGATTTCGACTACATGCACGGACGGCTCGCCAAGATTCCGGTAAAATACACGACCGGCGAAGCCGAACCGCCCCCCATTCTCGCAAACGATCTCCGCATCCCCCTCCTCCGCGAACTTCTCCAGAATATCAAGGTCGACACCGCACTCACCGATGGCGAAGCCGTCTCCTATGGTCTCCAACGCCGCACCATTCGCGGTTTCCGCGAGCTGCGCCGCATCTGGCGCGGCCATGCCCCCGCTGGCACGGTCTTTTCCAGCGAGTTGAAAGAAAAACTAGCCGGCGAACTCCGCCAGTTCCAGACCGACAACTCCACCACCATCGGCAGCGTCCAACTCTTCAACGAACTCATCCAAAACCCGAAATACTGGCCCATCTGGCAGGCTCCTGACGTCGAAACCGCCCGCCAATGGGCCGATGCCGGTTTTGCCGACGATCCGCTCGCCGCCCTTGTGCAAGAAGCCGAACTCCAGGAAGACATCGACGCCCTCAAGGCTCCAGTCAAACTCACTCCGGCCGATCCTGAGTATTCAAGAAGGCAATACGATTTCAATGCCGTCAGCAAATTCGGGGCCGGCTCCCGCTCCGCCAATCGCCACGAACCCGGGCAGACGGAGCGCGGCCACAACACCTTTACCACCGAAATCGCCGCCCGTAACGCGGCGGACGGGAACCGCTGGCGGGCAACCCACGTCCGCATCCATTACTCCGCTCCCCGCCTTCTTCGTGACGGACTCCGCCGACCTGACACCGACGGCAACGAAGCCCTGGAAGCCGTCCCTTGGCTCCAGCCCATGATGGAAGCCCTCGCCCCTCTCCCGACGCTTCCGCAAGACCTCACAGGCATGCCGGTCTTCCTCATGCCCGACGTCACCCTTTCCGGTGAGCGTCGCATCCTCCTCAATCTTCCTGTCACCCTCGAACCAGCCGCTCTTGTCGAACAACTGGGCAACGCCGGTCGCTGGCAAAACCAGTTCTTCGGCTCCCGCGAAGATCCATTCGCTCTCCGATGGCCCGCCGACGGTGCTGTAAAAACCGCCAAGGGGAAAACCCACATACCTTGGCACCAGGACCGCGATCACTTCACCGTACTCGGCGTGGATCTCGGCACGCGCGATGCCGGGGCGCTCGCTCTTCTCAACGTCACTGCGCAAAAACCGGCCAAGCCGGTCCACCGCATCATTGGTGAGGCCGACGGACGCACCTGGTATGCCAGCCTTGCCGACGCTCGCATGATCCGCCTGCCCGGGGAGGATGCCCGGCTCTTTGTCCGGGGAAAACTCGTTCAGGAACCCTATGGTGAACGCGGGCGAAACGCGTCTCTTCTCGAATGGGAAGACGCCCGCAATATCATCCTTCGCCTTGGCCAAAATCCCGACGAACTCCTCGGCGCCGATCCCCGGCGCCATTCGTATCCGGAAATAAACGATAAACTTCTCGTCGCCCTTCGCCGCGCTCAGGCCCGTCTTGCCCGTCTCCAGAACCGGAGCTGGCGGTTGCGCGACCTTGCAGAATCGGACAAGGCCCTTGATGAAATCCATGCCGAGCGTGCCGGGGAGAAGCCTTCTCCGCTTCCGCCCTTGGCTCGCGACGATGCCATCAAAAGCACCGACGAAGCCCTCCTTTCCCAGCGTGACATCATCCGGCGATCCTTCGTTCAGATCGCCAACTTGATCCTTCCCCTTCGCGGACGCCGATGGGAATGGCGGCCCCATGTCGAGGTCCCGGATTGCCACATCCTTGCGCAGAGCGATCCCGGTACGGATGACACCAAGCGTCTTGTCGCCGGACAACGCGGCATCTCTCACGAGCGTATCGAGCAAATCGAAGAACTCCGTCGTCGCTGCCAATCCCTCAACCGTGCCCTGCGTCACAAACCCGGAGAGCGTCCCGTGCTCGGACGCCCCGCCAAGGGCGAGGAAATCGCCGATCCCTGTCCCGCGCTCCTCGAAAAGATCAACCGTCTCCGGGACCAGCGCGTTGACCAAACCGCGCATGCCATCCTCGCCGCCGCTCTCGGTGTTCGACTCCGCGCCCCCTCAAAAGACCGCGCCGAACGCCGCCATCGCGACATCCATGGCGAATACGAACGCTTTCGTGCGCCCGCTGATTTTGTCGTCATCGAAAACCTCTCCCGTTATCTCAGCTCGCAGGATCGTGCTCGTAGTGAAAACACCCGTCTCATGCAGTGGTGCCATCGCCAGATCGTGCAAAAACTCCGTCAGCTCTGCGAGACCTACGGCATCCCCGTCCTCGCCGTCCCGGCGGCCTACTCATCGCGTTTTTCTTCCCGGGACGGCTCGGCCGGATTCCGGGCCGTCCATCTGACACCGGACCACCGTCACCGGATGCCATGGAGCCGCATCCTCGCCCGCCTCAAGGCCCACGAGGAAGACGGAAAAAGACTCGAAAAGACGGTGCTCGACGAGGCTCGCGCCGTCCGGGGACTCTTTGACCGGCTCGACCGGTTCAACGCCGGGCATGTCCCGGGAAAACCTTGGCGCACGCTCCTCGCGCCGCTCCCCGGCGGCCCTGTGTTTGTCCCCCTCGGGGACGCCACACCCATGCAGGCCGATCTGAACGCCGCCATCAACATCGCCCTCCGGGGCATCGCGGCTCCCGACCGCCACGACATCCATCACCGGCTCCGTGCCGAAAACAAAAAACGCATCCTGAGCTTGCGTCTCGGCACTCAGCGCGAGAAAGCCCGCTGGCCTGGAGGAGCTCCGGCGGTGACACTCTCCACTCCGAACAACGGCGCCTCTCCCGAAGATTCCGATGCGTTGCCCGAACGGGTATCCAACCTGTTTGTGGACATCGCCGGTGTCGCCAACTTCGAGCGAGTCACGATCGAAGGAGTCTCGCAAAAATTCGCCACCGGGCGTGGCCTTTGGGCCTCCGTCAAGCAACGTGCATGGAACCGCGTTGCCAGACTCAACGAGACAGTAACAGATAACAACAGGAACGAAGAGGAGGACGACATTCCGATGTAACCATTGCTTCATTACATCTGAGTCTCCCCTCAATCCCTCTGCCCCATGCGTGATATAACCTCCACCTCATGTCCCGGATCGGCGCCGGCAACCTGTAGTTCCCTTCCATCCTCCAACACTCCCGCAGATCGCGATCCGCTGCCGCCGATGCCGGTGCGCCGCCTTCACAACTATCTCTACTGTCCGCGGCTTTTTTATCTCCAGTGGGTCGAGAATCTCTTTGAGGAAAATGCCGACACCATTGCCGGCAGCGCCGTGCATCGTCACGCCGACAAACCTACGCGTTACGATGATGAAAAAGCCGAGGCACTTCGCACTGGTCTCCCTGAAGGCGCGCACATACGCAGCCTTCGCCTGGAAAACGCCCAACTCGGTCTCGTTGGCGTGGTGGATATCGTGGAGGGAGGCCCCGACGGACTCGAACTCGTCGACTACAAAAAAGGTTCCGCCTTCCGCCTCGACGACGGCACGCTCGCTCCCAAGGAAAACGACACCGTGCAACTTGCCGCCTACGCTCTTCTCCTGGCTGCCGATGGTGCGCGCGTTGCGCCCATGGCGACGGTCTATTACGCTGCCGATCGCCGGCGTGTCACCTTCCCGCTCGATGACGCCCTCTACGCCCGCACCCGTTCCGCCCTCGAAGAGGCCCGCGCCGTTGCAACCTCGGGGCGCATACCTCCGCCGCTCGTCTCTGACGTCCGCTGCCTCCATTGTTCCTCCTATGCGCTTTGCCTTCCCCGCGAGTCCGCCTGGTGGTGCCGCCATCGCAGCACGCCGCGGGGAGCCGGCCACACCCCCATGTTGCCGGGCTTTGAGGATGACGCCGCCGCCATTCACCAAATCTCCGAACCTGACACCGAGCCACCACCCGATCTTGCCAGCCAGCCTCCCCGTCCCCCGCGGCTCGATGGAGAATTGTTGGTTGTCCAGACTCCGGGAGCGATGATCGGACAAAGCGGCGGTGAGTTTACCGTGTCCGTCAAGGGTGAGGTTTTGCGCAAGCTTCCGGTTCATCAACTCCGGGCCATTTACGTTTACGGAGCCGTGCAACTCACGGCGCATGCTGTGCAGACCGCCCTTGAGGAGGATATCGACGTCTCCTATTTTGCGCCCAGCGGCCGCTTTCTTGGCCTCCTCCGCGGCCTGCCCGCATCCGGCGTGGATGCGCGTCTCGGGCAATACACCCTGTTTCGCGAACCCTTTGGCCGTCTCCGTCTCGCCTGCGAGGCGATTCGGGCCAAGATCCATAACCAGCGCGTCCTCCTCATGCGTAACGGCGAGCCCGGGGAGGGCGTCTTGCGCGAACTCGCCCGTCTGCGCGACGCCACCAGTGAGGCGACTTCGCTCGACGAACTCCTCGGCATCGAGGGCATCGCCGCGCATTTCTATTTCCAGTATTTTCCCACCATGCTGAAAGAACGGGCGGCCTGGGCCTTTGATTTTTCCGGACGCAATCGCCGCCCGCCGCGCGACCCGGTCAACGCCCTGCTTTCGTTCGGTTACAGCGTGTTGTCCAAGGAACTTGCCGGCGTCTGCCACGCTGTTGGCCTAGACCCGTTTTTCGGCTTCATGCACCAGCCGCGTTACGGGCGCCCCGCACTCGCTCTCGATCTGATGGAGGAGTTTCGCCCTCTCATCGCCGACAGTGTTGCCCTGAATCTCATCAACCGTGGCGAACTCGACGAAGGGGACTTTATCCGGTCGGCCAATGGCACCGCGCTCAATGATCGGGGCCGCCGGCGTTTTTGGGAGGCATGGTTCCGGCGTCTCGACAGCGAAGTCAGCCATCCTGAATTTGGTTACAAGATGAGCTATCGACGGATGCTTGAAGTGCAGGCGCGCCAGCTATGGCGCTATGTGCGCGGTGACGCCTTCCGCTACCACGGATTCACCACCCGTTGATTCCGATGTCAGATCCCCGCCGCCGTTATCTTGTGTGTTACGACATCGCCAATCCGAAGCGATTGCGCCAAGTGGCCAAGCTGCTGGAGAGCTATGGCACGCGTCTGCAATACTCGGTTTTCGAATGTCCTTTGGACGATCTTCGTCTTGAACAGGCGAAGGCTGATTTGCGCGACACGATTAATGCCGACCAAGACCAGGTGTTATTTGTTTCGCTTGGCCCCGAAGCCAACGATGCCACGTTGATCATCGCCACGCTTGGGCTCCCTTATACCGTGCGCTCGCGAGTGACGATTATCTGACCCATAACCCACGTGTTGAAGAGGCTGAAAACAGACGGACCTCTATGAAGAACAATTGACGTTTTGGCCGAACTCAGCAGACCTTTATGCGGCTAAGGCCAATGATCATCCATCCTACCGCCATTGGGCTGGAGACGTTTTTTGAAACGGCGAGTGCTGCGGATAGCGAGTTTCTCTTGGGGAGGCGCTCGCGGCCACTTTTACAGAGGAGATGTTCGGGCGAACTGGCCGACCTAACAAGGCGTACCCGGCTCAAAATCGAGGCACGCTCGCACGGGATGATGTAATTCGTTGTTTTTCAGCATACCGTGCGAGCACGGGCCGCAGCGAATGCCGTTTCACGAATCGTCAGGCGGCGGGGAGAAGTCATTTAATAAGGCCACTGTTAAAAGCCGCAGCGAATGCCGTTTCACGAATCGTCAGGCGGGCAGTGGATGTTTTTCCATGAGGCGAAGAATTTCATCGCCGCAGTGAATGCCGTTTCACCATTGATGAAGAATGCGAGGTGAAAACAGAGAAATTGGGTCAACTCTATCACTCTTATTCAGCCATCGTTTCAAGAAAGGATACCTCGTATTGGATACAACACAGCTCGTTCGTTCTCTCTACCTCCCTCGACAATCTCAAGGA >B-locus (SEQ ID NO: 73)TAATAAAATTGAAATATCACTATGGATTATTGTAATATTACCATAAAGATAGGTGACGTTTTTTTGAAAATTGTAAACCTAATTTGAAGAAAACCAATTAAAAATCGCTTCGGCTTTTTTTTAAGTGCCAGGTAGCATTGATGCTAACCCATGTGTAATAAAGGTTTGTTTTCCTTCGGGGCACGAACACATTATAAGGGAAACCTAAAGATTCCCTTTCTTGTTTAATATTATAACCAGTGAAAATAAGAATAATGCACCTAAAACTAATATACAGAAAATAAGAATTAAAAGTACTAATATATACATCATATGTTATCCTCCAATGCTTTATTTTTTAATAATTGATGTTAGTATTAGTTTTATTTTAATTTCTAAACATAAGAATTTGAAAAGGATGTGTTTATTATGGCGACACGCAGTTTTATTTTAAAAATTGAACCAAATGAAGAAGTTAAAAAGGGATTATGGAAGACGCATGAGGTATTGAATCATGGAATTGCCTACTACATGAATATTCTGAAACTAATTAGACAGGAAGCTATTTATGAACATCATGAACAAGATCCTAAAAATCCGAAAAAAGTTTCAAAAGCAGAAATACAAGCCGAGTTATGGGATTTTGTTTTAAAAATGCAAAAATGTAATAGTTTTACACATGAAGTTGACAAAGATGTTGTTTTTAACATCCTGCGTGAACTATATGAAGAGTTGGTCCCTAGTTCAGTCGAGAAAAAGGGTGAAGCCAATCAATTATCGAATAAGTTTCTGTACCCGCTAGTTGATCCGAACAGTCAAAGTGGGAAAGGGACGGCATCATCCGGACGTAAACCTCGGTGGTATAATTTAAAAATAGCAGGCGACCCATCGTGGGAGGAAGAAAAGAAAAAATGGGAAGAGGATAAAAAGAAAGATCCCCTTGCTAAAATCTTAGGTAAGTTAGCAGAATATGGGCTTATTCCGCTATTTATTCCATTTACTGACAGCAACGAACCAATTGTAAAAGAAATTAAATGGATGGAAAAAAGTCGTAATCAAAGTGTCCGGCGACTTGATAAGGATATGTTTATCCAAGCATTAGAGCGTTTTCTTTCATGGGAAAGCTGGAACCTTAAAGTAAAGGAAGAGTATGAAAAAGTTGAAAAGGAACACAAAACACTAGAGGAAAGGATAAAAGAGGACATTCAAGCATTTAAATCCCTTGAACAATATGAAAAAGAACGGCAGGAGCAACTTCTTAGAGATACATTGAATACAAATGAATACCGATTAAGCAAAAGAGGATTACGTGGTTGGCGTGAAATTATCCAAAAATGGCTAAAGATGGATGAAAATGAACCATCAGAAAAATATTTAGAAGTATTTAAAGATTATCAACGGAAACATCCACGAGAAGCCGGGGACTATTCTGTCTATGAATTTTTAAGCAAGAAAGAAAATCATTTTATTTGGCGAAATCATCCTGAATATCCTTATTTGTATGCTACATTTTGTGAAATTGACAAAAAAAAGAAAGACGCTAAGCAACAGGCAACTTTTACTTTGGCTGACCCGATTAACCATCCGTTATGGGTACGATTTGAAGAAAGAAGCGGTTCGAACTTAAACAAATATCGAATTTTAACAGAGCAATTACACACTGAAAAGTTAAAAAAGAAATTAACAGTTCAACTTGATCGTTTAATTTATCCAACTGAATCCGGCGGTTGGGAGGAAAAAGGTAAAGTAGATATCGTTTTGTTGCCGTCAAGACAATTTTATAATCAAATCTTCCTTGATATAGAAGAAAAGGGGAAACATGCTTTTACTTATAAGGATGAAAGTATTAAATTCCCCCTTAAAGGTACACTTGGTGGTGCAAGAGTGCAGTTTGACCGTGACCATTTGCGGAGATATCCGCATAAAGTAGAATCAGGAAATGTTGGACGGATTTATTTTAACATGACAGTAAATATTGAACCAACTGAGAGCCCTGTTAGTAAGTCTTTGAAAATACATAGGGACGATTTCCCCAAGTTCGTTAATTTTAAACCGAAAGAGCTCACCGAATGGATAAAAGATAGTAAAGGGAAAAAATTAAAAAGTGGTATAGAATCCCTTGAAATTGGTCTACGGGTGATGAGTATCGACTTAGGTCAACGTCAAGCGGCTGCTGCATCGATTTTTGAAGTAGTTGATCAGAAACCGGATATTGAAGGGAAGTTATTTTTTCCAATCAAAGGAACTGAGCTTTATGCTGTTCACCGGGCAAGTTTTAACATTAAATTACCGGGTGAAACATTAGTAAAATCACGGGAAGTATTGCGGAAAGCTCGGGAGGACAACTTAAAATTAATGAATCAAAAGTTAAACTTTCTAAGAAATGTTCTACATTTCCAACAGTTTGAAGATATCACAGAAAGAGAGAAGCGTGTAACTAAATGGATTTCTAGACAAGAAAATAGTGATGTTCCTCTTGTATATCAAGATGAGCTAATTCAAATTCGTGAATTAATGTATAAACCCTATAAAGATTGGGTTGCCTTTTTAAAACAACTCCATAAACGGCTAGAAGTCGAGATTGGCAAAGAGGTTAAGCATTGGCGAAAATCATTAAGTGACGGGAGAAAAGGTCTTTACGGAATCTCCCTAAAAAATATTGATGAAATTGATCGAACAAGGAAATTCCTTTTAAGATGGAGCTTACGTCCAACAGAACCTGGGGAAGTAAGACGCTTGGAACCAGGACAGCGTTTTGCGATTGATCAATTAAACCACCTAAATGCATTAAAAGAAGATCGATTAAAAAAGATGGCAAATACGATTATCATGCATGCCTTAGGTTACTGTTATGATGTAAGAAAGAAAAAGTGGCAGGCAAAAAATCCAGCATGTCAAATTATTTTATTTGAAGATTTATCTAACTACAATCCTTACGAGGAAAGGTCCCGTTTTGAAAACTCAAAACTGATGAAGTGGTCACGGAGAGAAATTCCACGACAAGTCGCCTTACAAGGTGAAATTTACGGATTACAAGTTGGGGAAGTAGGTGCCCAATTCAGTTCAAGATTCCATGCGAAAACCGGGTCGCCGGGAATTCGTTGCAGTGTTGTAACGAAAGAAAAATTGCAGGATAATCGCTTTTTTAAAAATTTACAAAGAGAAGGACGACTTACTCTTGATAAAATCGCAGTTTTAAAAGAAGGAGACTTATATCCAGATAAAGGTGGAGAAAAGTTTATTTCTTTATCAAAGGATCGAAAGTTGGTAACTACGCATGCTGATATTAACGCGGCCCAAAATTTACAGAAGCGTTTTTGGACAAGAACACATGGATTTTATAAAGTTTACTGCAAAGCCTATCAGGTTGATGGACAAACTGTTTATATTCCGGAGAGCAAGGACCAAAAACAAAAAATAATTGAAGAATTTGGGGAAGGCTATTTTATTTTAAAAGATGGTGTATATGAATGGGGTAATGCGGGGAAACTAAAAATTAAAAAAGGTTCCTCTAAACAATCATCGAGTGAATTAGTAGATTCGGACATACTGAAAGATTCATTTGATTTAGCAAGTGAACTTAAGGGAGAGAAACTCATGTTATATCGAGATCCGAGTGGAAACGTATTTCCTTCCGACAAGTGGATGGCAGCAGGAGTATTTTTTGGCAAATTAGAAAGAATATTGATTTCTAAGTTAACAAATCAATACTCAATATCAACAATAGAAGATGATTCTTCAAAACAATCAATGTAAAAGTTTGCCCGTATAAGAACTTAATTAATTAGGATGGTAGGATGTTACTAAATATGTCTGTAGGCATCATTCCTACTATCCGTTTTGTCCGAATATCAGAGCATTAGGTGAGGAATGGTAAGAAAGGAAAATTTATATGAACCAACCGATTCCTATTCGAATGTTAAATGAAATACAATATTGTGAGCGACTTTTTTACTTTATGCATGTCCAAAAGCTATTTGATGAGAATGCAGATACAGTTGAAGGAAGTGCACAGCATGAGCGGGCAGAAAGAAGCAAAAGACCAAGTAAAATGGGACCAAAGGAATTATGGGGTGAGGCGCCAAGAAGTCTTAAGCTTGGTGATGAGCTGTTAAATATTACCGGTGTTCTTGATGCCATAAGTCATGAAGAGAACAGTTGGATCCCGGTTGAATCAAAACACAGTTCCGCACCGGATGGATTGAACCCTTTTAAAGTAGATGGCTTTCTACTTGACGGGTCTGCATGGCCAAACGATCAAATTCAACTTTGTGCACAAGGCTTGCTCTTGAATGCCAATGGATACCCGTGTGATTATGGGTATTTATTTTATCGTGGTAATAAGAAAAAGGTGAAAATTTATTTTACTGAAGATTTAATCGCTGCCACAAAGTACTATATTAAAAAAGCACACGAGATACTAGTATTATCTGGTGATGAATCAGCTATTCCTAAGCCTTTAATTGATTCTAATAAGTGTTTTCGCTGTTCTTTAAACTATATCTGTCTTCCGGATGAAACGAACTATCTATTAGGGGCAAGTTCAACAATTCGTAAAATTGTGCCTTCAAGGACAGATGGTGGCGTTTTATATGTATCAGAGTCTGGTACAAAATTAGGAAAATCGGGTGAGGAGTTAATCATTCAGTATAAAGATGGCCAAAAGCAGGGTGTTCCTATAAAAGATATTATTCAAGTTTCGTTAATTGGAAATGTTCAATGCTCAACGCAATTACTTCATTTTTTAATGCAATCAAATATTCCTGTAAGTTATTTATCATCCCACGGTCGTTTGATTGGTGTCAGTTCATCTTTAGTTACAAAAAATGTTTTAACAAGGCAGCAACAGTTCATTAAATTTACAAATCCTGAGTTTGGACTAAATCTAGCAAAACAAATTGTTTATGCCAAGATTCGAAATCAACGAACTTTACTTAGAAGAAATGGGGGGAGTGAGGTAAAGGAGATTTTAACAGATTTAAAATCTTTAAGTGACAGTGCACTGAACGCAATATCAATAGAACAATTACGGGGTATTGAAGGGATTTCTGCAAAACATTATTTCGCAGGATTTCCGTTTATGTTGAAAAATGAATTACGTGAATTGAATTTAATGAAAGGGCGTAATAGGAGACCGCCAAAAGATCCTGTAAATGTACTTCTTTCTCTTGGTTATACTTTATTGACACGTGATATTCATGCTGCGTGTGGTTCAGTCGGATTGGATCCGATGTTTGGTTGTTACCATCGTCCAGAAGCAGGTCGACCGGCTCTAGTATTAGATGTTATGGAAACATTTCGACCACTTATTGTAGACAGTATTGTCATCCGAGCTTTGAATACGGGTGAAATCTCATTAAAAGATTTTTATATAGGAAAAGATAGTTGTCAATTATTAAAACATGGCCGCGATTCCTTTTTTGCCATTTATGAAAGAAGAATGCATGAAACTATTACCGATCCAATTTTCGGCTATAAGATTAGCTATCGCCGTATGCTCGATTTGCACATTCGAATGCTTGCAAGGTTTATTGAAGGGGAACTGCCGGAATATAAACCATTAATGACCCGGTGAGTTTGTTTATTAGGTTAAAAGAAGGTGAAGACATGCAGCAATACGTCCTTGTTTCTTATGATATTTCGGACCAAAAAAGATGGAGAAAAGTATTTAAACTGATGAAAGGATACGGAGAACATGTTCAATATTCCGTATTCATATGCCAGTTAACTGAATTACAGAAGGCAAAATTACAAGCCTCTTTAGAAGACATTATCCATCATAAGAATGACCAAGTAATGTTTGTTCACATCGGGCCAGTGAAAGATGGTCAACTATCTAAAAAAATCTCAACAATTGGGAAAGAATTTGTTCCATTGGATTTAAAGCGGCTTATATTTTGAAAAGATATAGCAAAGAAATCTTATGAAAAAAATACAAAAATATATTGTTAAAAAATAGGGAATATTATATAATGGACTTACGAGGTTCTGTCTTTTGGTCAGGACAACCGTCTAGCTATAAGTGCTGCAGGGGTGTGAGAAACTCCTATTGCTGGACGATGTCTCTTTTATTTCTTTTTTCTTGGATCTGAGTACGAGCACCCACATTGGACATTTCGCATGGTGGGTGCTCGTACTATAGGTAAAACAAACCTTTTTAAGAAGAATACAAAAATAACCACAATATTTTTTAAAAGGAATTTTGATGGATTTACATAACCTCTCGCAACATGCTTCTAAAACCCAAGCCCACCATAGCCCAAAACCCCCTGCGGTCCAAGAAAAAAGAAATGATACGAGGCATTAGCACCGGGGAGAAGTCATTTAATAAGGCCACTGTTAAAAGTCCAAGAAAAAAGAAATGATACGAGGCATTAGCACAACAATATAAACGACTACTTTACCGTGTTCAAGAAAAAAGAAATGATATGAGGCATTAGCACGATGGGATGGGAGAGAGAGGACAGTTCTACTCTTGCTGTATCCAGCTTCTTTTACTTTATCCGGTATCATTTCTTCACTTCTTTCTGCACATAAAAAAGCACCTAACTATTTGGATAAGTTAAGTGCTTTTATTTCCGTTTGAAGTTGTCTATTGCTTTTTTCTTCATATCTTCAAATTTTTTCTGTTTCTCAGAGTCAACTTTACCAACTGTAATCCCTTTTCTTTTTGGCATTGGGGTATCTTTCCACCTTAGTGTGTTCATAAGGCTTATATTTATCACTCATTGTATTCCTCCAACACAATTATAATTTTTCCGTCATCCTCAATCCAACCGTCAACTGTGACAAAAGACGAATCTCTCTTAT >C-locus (SEQ ID NO: 74)GTTTCATTTGGAAAGGGAGAGCATTGGCTTTTCTCTTTGTAAATAAAGTGCAAGCTTTGTAATAAGCTTCTAGTGGAGAAGTGATTGTTTGAATCACCCAATGCACACGCACTAAAGTTAGACGAACCTATAATTCGTATTAGTAAGTATAGTACATGAAGAAAAATGCAACAAGCATTTACTCTCTTTTAAATAAAGAATTGATAGCTGTTAATATTGATAGTATATTATACCTTATAGATGTTCGATTTTTTTTGAAATTCAAAAATCATACTTAGTAAAGAAAGGAAATAACGTCATGGACAAGCGAAAGCGTAGAAGTTACGAGTTTAGGTGGGAAGCGGGAGGCACCAGTCATGGCAATCCGTAGCATAAAACTAAAACTAAAAACCCACACAGGCCCGGAAGCGCAAAACCTCCGAAAAGGAATATGGCGGACGCATCGGTTGTTAAATGAAGGCGTCGCCTATTACATGAAAATGCTCCTGCTCTTTCGTCAGGAAAGCACTGGTGAACGGCCAAAAGAAGAACTACAGGAAGAACTGATTTGTCACATACGCGAACAGCAACAACGAAATCAGGCAGATAAAAATACGCAAGCGCTTCCGCTAGATAAGGCACTGGAAGCTTTGCGCCAACTATATGAACTGCTTGTCCCCTCCTCGGTCGGACAAAGTGGCGACGCCCAGATCATCAGCCGAAAGTTTCTCAGCCCGCTCGTCGATCCGAACAGCGAAGGCGGCAAAGGTACTTCGAAGGCAGGGGCAAAACCCACTTGGCAGAAGAAAAAAGAAGCGAACGACCCAACCTGGGAACAGGATTACGAAAAATGGAAAAAAAGACGCGAGGAAGACCCAACCGCTTCTGTGATTACTACTTTGGAGGAATACGGCATTAGACCGATCTTTCCCCTGTACACGAACACCGTAACAGATATCGCGTGGTTGCCACTTCAATCCAATCAGTTTGTGCGAACCTGGGACAGAGACATGCTTCAACAAGCGATTGAAAGACTGCTCAGTTGGGAGAGCTGGAACAAACGTGTCCAGGAAGAGTATGCCAAGCTGAAAGAAAAAATGGCTCAACTGAACGAGCAACTCGAAGGCGGTCAGGAATGGATCAGCTTGCTAGAGCAGTACGAAGAAAACCGAGAGCGAGAGCTTAGGGAAAACATGACCGCTGCCAATGACAAGTATCGGATTACCAAGCGGCAAATGAAAGGCTGGAACGAGCTGTACGAGCTATGGTCAACCTTTCCCGCCAGTGCCAGTCACGAGCAATACAAAGAGGCGCTCAAGCGTGTGCAGCAGCGACTGAGAGGGCGGTTTGGGGATGCTCATTTCTTCCAGTATCTGATGGAAGAGAAGAACCGCCTGATCTGGAAGGGGAATCCGCAGCGTATCCATTATTTTGTCGCGCGCAACGAACTGACGAAACGGCTGGAGGAAGCCAAGCAAAGCGCCACGATGACGTTGCCCAATGCCAGGAAGCATCCATTGTGGGTGCGCTTCGATGCACGGGGAGGAAATTTGCAAGACTACTACTTGACGGCTGAAGCGGACAAACCGAGAAGCAGACGTTTTGTAACGTTTAGTCAGTTGATATGGCCAAGCGAATCGGGATGGATGGAAAAGAAAGACGTCGAGGTCGAGCTAGCTTTGTCCAGGCAGTTTTACCAGCAGGTGAAGTTGCTGAAAAATGACAAAGGCAAGCAGAAAATCGAGTTCAAGGATAAAGGTTCGGGCTCGACGTTTAACGGACACTTGGGGGGAGCAAAGCTACAACTGGAGCGGGGCGATTTGGAGAAGGAAGAAAAAAACTTCGAGGACGGGGAAATCGGCAGCGTTTACCTTAACGTTGTCATTGATTTCGAACCTTTGCAAGAAGTGAAAAATGGCCGCGTGCAGGCGCCGTATGGACAAGTACTGCAACTCATTCGTCGCCCCAACGAGTTTCCCAAGGTCACTACCTATAAGTCGGAGCAACTTGTTGAATGGATAAAAGCTTCGCCACAACACTCGGCTGGGGTGGAGTCGCTGGCATCCGGTTTTCGTGTAATGAGCATAGACCTTGGGCTGCGCGCGGCTGCAGCGACTTCTATTTTTTCTGTAGAAGAGAGTAGCGATAAAAATGCGGCTGATTTTTCCTACTGGATTGAAGGAACGCCGCTGGTCGCTGTCCATCAGCGGAGCTATATGCTCAGGTTGCCTGGTGAACAGGTAGAAAAACAGGTGATGGAAAAACGGGACGAGCGGTTCCAGCTACACCAACGTGTGAAGTTTCAAATCAGAGTGCTCGCCCAAATCATGCGTATGGCAAATAAGCAGTATGGAGATCGCTGGGATGAACTCGACAGCCTGAAACAAGCGGTTGAGCAGAAAAAGTCGCCGCTCGATCAAACAGACCGGACATTTTGGGAGGGGATTGTCTGCGACTTAACAAAGGTTTTGCCTCGAAACGAAGCGGACTGGGAACAAGCGGTAGTGCAAATACACCGAAAAGCAGAGGAATACGTCGGAAAAGCCGTTCAGGCATGGCGCAAGCGCTTTGCTGCTGACGAGCGAAAAGGCATCGCAGGTCTGAGCATGTGGAACATAGAAGAATTGGAGGGCTTGCGCAAGCTGTTGATTTCCTGGAGCCGCAGGACGAGGAATCCGCAGGAGGTTAATCGCTTTGAGCGAGGCCATACCAGCCACCAGCGTCTGTTGACCCATATCCAAAACGTCAAAGAGGATCGCCTGAAGCAGTTAAGTCACGCCATTGTCATGACTGCCTTGGGGTATGTTTACGACGAGCGGAAACAAGAGTGGTGCGCCGAATACCCGGCTTGCCAGGTCATTCTGTTTGAAAATCTGAGCCAGTACCGTTCTAACCTGGATCGCTCGACCAAAGAAAACTCCACCTTGATGAAGTGGGCGCATCGCAGCATTCCGAAATACGTCCACATGCAGGCGGAGCCATACGGGATTCAGATTGGCGATGTCCGGGCGGAATATTCCTCTCGTTTTTACGCCAAGACAGGAACGCCAGGCATTCGTTGTAAAAAGGTGAGAGGCCAAGACCTGCAGGGCAGACGGTTTGAGAACTTGCAGAAGAGGTTAGTCAACGAGCAATTTTTGACGGAAGAACAAGTGAAACAGCTAAGGCCCGGCGACATTGTCCCGGATGATAGCGGAGAACTGTTCATGACCTTGACAGACGGAAGCGGAAGCAAGGAGGTCGTGTTTCTCCAGGCCGATATTAACGCGGCGCACAATCTGCAAAAACGTTTTTGGCAGCGATACAATGAACTGTTCAAGGTTAGCTGCCGCGTCATCGTCCGAGACGAGGAAGAGTATCTCGTTCCCAAGACAAAATCGGTGCAGGCAAAGCTGGGCAAAGGGCTTTTTGTGAAAAAATCGGATACAGCCTGGAAAGATGTATATGTGTGGGACAGCCAGGCAAAGCTTAAAGGTAAAACAACCTTTACAGAAGAGTCTGAGTCGCCCGAACAACTGGAAGACTTTCAGGAGATCATCGAGGAAGCAGAAGAGGCGAAAGGAACATACCGTACACTGTTCCGCGATCCTAGCGGAGTCTTTTTTCCCGAATCCGTATGGTATCCCCAAAAAGATTTTTGGGGCGAGGTGAAAAGGAAGCTGTACGGAAAATTGCGGGAACGGTTTTTGACAAAGGCTCGGTAAGGGTGTGCAAGGAGAGTGAATGGCTTGTCCTGGATACCTGTCCGCATGCTAAATGAAATTCAGTATTGTGAGCGACTGTACCATATTATGCATGTGCAGGGGCTGTTTGAGGAAAGCGCAGACACGGTCGAAGGAGCAGCACAACACAAGCGTGCAGAGACACATCTGCGCAAAAGCAAGGCAGCGCCGGAAGAGATGTGGGGGGACGCTCCGTTTAGCTTGCAGCTCGGCGACCCTGTGCTTGGCATTACGGGAAAGCTGGATGCCGTCTGTCTGGAAGAAGGTAAGCAGTGGATTCCGGTAGAAGGAAAGCATTCGGCGTCGCCAGAAGGCGGGCAGATGTTCACTGTAGGCGTGTATTCGCTGGACGGTTCTGCCTGGCCCAACGACCAAATCCAATTGTGTGCGCAAGGCTTGCTGCTTCGCGCGAATGGATATGAATCCGATTATGGCTACTTATACTACCGTGGCAATAAAAAGAAGGTTCGCATTCCTTTTTCGCAGGAACTCATAGCGGCTACTCACGCCTGCATTCAAAAAGCTCATCAGCTTCGGGAAGCCGAAATTCCCCCTCCGTTGCAGGAGTCGAAAAAGTGCTTTCGATGCTCGTTAAATTACGTATGCATGCCTGACGAGACGAATTACATGTTGGGGTTGAGCGCAAACATCAGAAAGATTGTGCCCAGTCGTCCAGATGGCGGGGTACTGTATGTTACAGAGCAGGGGGCAAAACTGGGCAGAAGCGGAGAAAGCTTGACCATCACCTGCCGGGGCGAAAAGATAGACGAAATCCCGATCAAAGACTTGATTCACGTGAGCTTGATGGGGCATGTGCAATGCTCTACGCAGCTTCTGCACACCTTGATGAACTGTGGCGTCCACGTCAGCTACTTGACTACGCATGGCACATTGACAGGAATAATGACTCCCCCTTTATCGAAAAACATTCGAACAAGAGCCAAGCAGTTTATCAAATTTCAGCACGCGGAGATCGCCCTTGGAATCGCGAGAAGGGTCGTGTATGCGAAAATTTCCAATCAGCGCACGATGCTGCGCCGCAATGGCTCACCAGATAAAGCAGTTTTAAAAGAGTTAAAAGAGCTTAGAGATCGCGCGTGGGAGGCGCCATCACTGGAAATAGTGAGAGGTATCGAGGGACGTGCAGCACAGTTGTACATGCAGTTTTTCCCTACCATGTTAAAGCACCCAGTAGTAGACGGTATGGCGATCATGAACGGTCGCAACCGTCGCCCGCCCAAAGATCCGGTCAATGCGCTGCTCTCCCTCGGCTATACGCTTCTTTCACGGGATGTTTACTCCGCATGTGCCAATGTCGGACTCGATCCACTGTTCGGCTTTTTCCATACGATGGAGCCGGGCAGACCAGCTTTGGCACTCGATCTGATGGAACCGTTCCGCGCCTTGATTGCCGATAGCGTAGCGATACGTACCTTGAATACGGAGGAACTCACCCTCGGGGACTTTTATTGGGGAAAAGACAGTTGTTATTTGAAAAAGGCAGGAAGACAAACGTATTTCGCTGCCTATGAAAGACGGATGAACGAGACGCTGACGCATCCGCAATTTGGGTATAAGCTCAGCTATCGCCGTATGCTGGAGCTGGAAGCAAGGTTTTTGGCCCGGTATCTGGATGGAGAGCTGGTGGAATATACGCCGCTCATGACAAGGTAGGAAATGACCATGCGACAATTTGTTCTGGTAAGCTATGATATTGCCGATCAAAAACGTTGGAGAAAAGTATTCAAGCTGATGAAGGGGCAAGGCGAGCACGTCCAGTACTCGGTGTTTCTGTGCCAACTCACCGAGATTCAGCAAGCCAAGCTAAAGGTAAGCCTGGCGGAGCTGGTTCACCATGGAGAAGACCAGGTCATGTTTGTAAAAATCGGCCCAGTGACGAGAGATCAACTGGACAAGCGGATATCTACTGTTGGCAGGGAGTTTCTGCCTCGCGATTTGACCAAATTTATCTATTAAGGAATGAAGAAAGCTAGTTGTAACAAAAGTGGAAAAAGAGTAAAATAAAGGTGTCAGTCGCACGCTATAGGCCATAAGTCGACTTACATATCCGTGCGTGTGCATTATGGGCCCATCCACAGGTCTATTCCCACGGATAATCACGACTTTCCACTAAGCTTTCGAATTTTATGATGCGAGCATCCTCTCAGGTCAAAAAAGCCGGGGGATGCTCGAACTCTTTGTGGGCGTAGGCTTTCCAGAGTTTTTTAGGGGAAGAGGCAGCCGATGGATAAGAGGAATGGCGATTGAATTTTGGCTTGCTCGAAAAACGGGTCTGTAAGGCTTGCGGCTGTAGGGGTTGAGTGGGAAGGAGTTCGAAAGCTTAGTGGAAAGCTTCGTGGTTAGCACCGGGGAGAAGTCATTTAATAAGGCCACTGTTAAAAGTTCGAAAGCTTAGTGGAAAGCTTCGTGGTTAGCACGCTAAAGTCCGTCTAAACTACTGAGATCTTAAATCGGCGCTCAAATAAAAAACCTCGCTAATGCGAGGTTTCAGC >D-locus (SEQ ID NO: 75)GAAGTTATGTTGATAAAATGGTTTATGAAAACGTGAGTCTGTGGTAGTATTATAAACAATGATGGAATAAAGTGTTTTTTGCGCCGCACGGCATGAATTCAGGGGTTAGCTTGGTTTTGTGTATAAATAAATGTTCTACATATTTATTTTGTTTTTTGCGCCGCAAAATGCAACTGAAAGCCGCATCTAGAGCACCCTGTAGAAGACAGGGTTTTGAGAATAGCCCGACATAGAGGGCAATAGACACGGGGAGAAGTCATTTAATAAGGCCACTGTTAAAAGTTTTGAGAATAGCCCGACATAGAGGGCAATAGACTTTTGCTTCGTCACGGATGGACTTCACAATGGCAACAACGTTTTGAGAATAGCCCGACATAGTTATAGAGATGTATAAATATAACCGATAAACATTGACTAATTTGTTGAAGTCAGTGTTTATCGGTTTTTTGTGTAAATATAGGAGTTGTTAGAATGATACTTTTTGCCTAATTTTGGAACTTTATGAGGATATAAGATAGACTTGATAAAAAGGTAAAAGAAAGGTTAAAGAGCATGGCAGGAATAGTGACCTGTGATGAAGATGATGGTAGAATTAAAAGTGTTCTTAAAGAAAAACAATATTGGATAAGGAAAATAATTCAATAGATAAAAAATTTAGGGGGAAAAATGAAAATATCAAAAGTCGATCATACCAGAATGGCGGTTGCTAAAGGTAATCAACACAGGAGAGATGAGATTAGTGGGATTCTCTATAAGGATCCGACAAAGACAGGAAGTATAGATTTTGATGAACGATTCAAAAAACTGAATTGTTCGGCGAAGATACTTTATCATGTATTCAATGGAATTGCTGAGGGAAGCAATAAATACAAAAATATTGTTGATAAAGTAAATAACAATTTAGATAGGGTCTTATTTACAGGTAAGAGCTATGATCGAAAATCTATCATAGACATAGATACTGTTCTTAGAAATGTTGAGAAAATTAATGCATTTGATCGAATTTCAACAGAGGAAAGAGAACAAATAATTGACGATTTGTTAGAAATACAATTGAGGAAGGGGTTAAGGAAAGGAAAAGCTGGATTAAGAGAGGTATTACTAATTGGTGCTGGTGTAATAGTTAGAACCGATAAGAAGCAGGAAATAGCTGATTTTCTGGAGATTTTAGATGAAGATTTCAATAAGACGAATCAGGCTAAGAACATAAAATTGTCTATTGAGAATCAGGGGTTGGTGGTCTCGCCTGTATCAAGGGGAGAGGAACGGATTTTTGATGTCAGTGGCGCACAAAAGGGAAAAAGCAGCAAAAAAGCGCAGGAGAAAGAGGCACTATCTGCATTTCTGTTAGATTATGCTGATCTTGATAAGAATGTCAGGTTTGAGTATTTACGTAAAATTAGAAGACTGATAAATCTATATTTCTATGTCAAAAATGATGATGTTATGTCTTTAACTGAAATTCCGGCAGAAGTGAATCTGGAAAAAGATTTTGATATCTGGAGAGATCACGAACAAAGAAAGGAAGAGAATGGAGATTTTGTTGGATGTCCGGACATACTTTTGGCAGATCGTGATGTGAAGAAAAGTAACAGTAAGCAGGTAAAAATTGCAGAGAGGCAATTAAGGGAGTCAATACGTGAAAAAAATATAAAACGATATAGATTTAGCATAAAAACGATTGAAAAGGATGATGGAACATACTTTTTTGCAAATAAGCAGATAAGTGTATTTTGGATTCATCGCATTGAAAATGCTGTAGAACGTATATTAGGATCTATTAATGATAAAAAACTGTATAGATTACGTTTAGGATATCTAGGAGAAAAAGTATGGAAGGACATACTCAATTTTCTCAGCATAAAATACATTGCAGTAGGCAAGGCAGTATTCAATTTTGCAATGGATGATCTGCAGGAGAAGGATAGAGATATAGAACCCGGCAAGATATCAGAAAATGCAGTAAATGGATTGACTTCGTTTGATTATGAGCAAATAAAGGCAGATGAGATGCTGCAGAGAGAAGTTGCTGTTAATGTAGCATTCGCAGCAAATAATCTTGCTAGAGTAACTGTAGATATTCCGCAAAATGGAGAAAAAGAGGATATCCTTCTTTGGAATAAAAGTGACATAAAAAAATACAAAAAGAATTCAAAGAAAGGTATTCTGAAATCTATACTTCAGTTTTTTGGTGGTGCTTCAACTTGGAATATGAAAATGTTTGAGATTGCATATCATGATCAGCCAGGTGATTACGAAGAAAACTACCTATATGACATTATTCAGATCATTTACTCGCTCAGAAATAAGAGCTTTCATTTCAAGACATATGATCATGGGGATAAGAATTGGAATAGAGAACTGATAGGAAAGATGATTGAGCATGATGCTGAAAGAGTCATTTCTGTTGAGAGGGAAAAGTTTCATTCCAATAACCTGCCGATGTTTTATAAAGACGCTGATCTAAAGAAAATATTGGATCTCTTGTATAGCGATTATGCAGGACGTGCATCTCAGGTTCCGGCATTTAACACTGTCTTGGTTCGAAAGAACTTTCCGGAATTTCTTAGGAAAGATATGGGCTACAAGGTTCATTTTAACAATCCTGAAGTAGAGAATCAGTGGCACAGTGCGGTGTATTACCTATATAAAGAGATTTATTACAATCTATTTTTGAGAGATAAAGAGGTAAAGAATCTTTTTTATACTTCATTAAAAAATATAAGAAGTGAAGTTTCGGACAAAAAACAAAAGTTAGCTTCAGATGATTTTGCATCCAGGTGTGAAGAAATAGAGGATAGAAGTCTTCCGGAAATTTGTCAGATAATAATGACAGAATACAATGCGCAGAACTTTGGTAATAGAAAAGTTAAATCTCAGCGTGTTATTGAAAAAAATAAGGATATTTTCAGACATTATAAAATGCTTTTGATAAAGACTTTAGCAGGTGCTTTTTCTCTTTATTTGAAGCAGGAAAGATTTGCATTTATTGGTAAGGCAACACCTATACCATACGAAACAACCGATGTTAAGAATTTTTTGCCTGAATGGAAATCCGGAATGTATGCATCGTTTGTAGAGGAGATAAAGAATAATCTTGATCTTCAAGAATGGTATATCGTCGGACGATTCCTTAATGGGAGGATGCTCAATCAATTGGCAGGAAGCCTGCGGTCATACATACAGTATGCGGAAGATATAGAACGTCGTGCTGCAGAAAATAGGAATAAGCTTTTCTCCAAGCCTGATGAAAAGATTGAAGCATGTAAAAAAGCGGTCAGAGTGCTTGATTTGTGTATAAAAATTTCAACTAGAATATCTGCGGAATTTACTGACTATTTTGATAGTGAAGATGATTATGCAGATTATCTTGAAAAATATCTCAAGTATCAGGATGATGCCATTAAGGAATTGTCAGGATCTTCGTATGCTGCGTTGGATCATTTTTGCAACAAGGATGATCTGAAATTTGATATCTATGTAAATGCCGGACAGAAGCCTATCTTACAGAGAAATATCGTGATGGCAAAGCTTTTTGGACCAGATAACATTTTGTCTGAAGTTATGGAAAAGGTAACAGAAAGTGCCATACGAGAATACTATGACTATCTGAAGAAAGTTTCAGGATATCGGGTAAGGGGAAAATGTAGTACAGAGAAAGAACAGGAAGATCTGCTAAAGTTCCAAAGATTGAAAAACGCAGTAGAATTCCGGGATGTTACTGAATATGCTGAGGTTATTAATGAGCTTTTAGGACAGTTGATAAGTTGGTCATATCTTAGGGAGAGGGATCTATTATATTTCCAGCTGGGATTCCATTACATGTGTCTGAAAAACAAATCTTTCAAACCGGCAGAATATGTGGATATTCGTAGAAATAATGGTACGATTATACATAATGCGATACTTTACCAGATTGTTTCGATGTATATTAATGGACTGGATTTCTATAGTTGTGATAAAGAAGGGAAAACGCTCAAACCAATTGAAACAGGAAAGGGCGTAGGAAGTAAGATAGGACAATTTATAAAGTATTCCCAGTATTTATACAATGATCCGTCATATAAGCTTGAGATCTATAATGCAGGATTAGAAGTTTTTGAAAACATTGATGAACATGATAATATTACAGATCTTAGAAAGTATGTGGATCATTTTAAGTATTATGCATATGGTAATAAAATGAGCCTGCTTGATCTGTATAGTGAATTCTTCGATCGTTTCTTTACATATGATATGAAGTATCAGAAGAATGTAGTGAATGTGTTGGAGAATATCCTTTTAAGGCATTTTGTAATTTTCTATCCGAAGTTTGGATCAGGAAAAAAAGATGTTGGAATTAGGGATTGTAAAAAAGAAAGAGCTCAGATTGAAATAAGTGAGCAGAGCCTCACATCGGAAGACTTCATGTTTAAGCTTGACGACAAAGCAGGAGAAGAAGCAAAGAAGTTTCCGGCAAGGGATGAACGTTATCTCCAGACAATAGCCAAGTTGCTCTATTATCCTAACGAAATTGAGGATATGAACAGATTCATGAAGAAAGGAGAAACGATAAATAAAAAAGTTCAGTTTAATAGAAAAAAGAAGATAACCAGGAAACAAAAGAATAATTCATCAAACGAGGTATTGTCTTCAACTATGGGTTATTTATTTAAGAACATTAAATTGTAAAAAAGATTCGTTGTAGATAATTGATAGGTAAAAGCTGACCGGAGCCTTTGGCTCCGGACAGTTGTATATAAGAGGATATTAATGACTGAAAATGATTTTTGTTGGAAGTCAGTTTTTTCTGTGGAAAGCGAAATCGAATATGATGAGTATGCATATGGCAGAAGAGCTGTAGAAGGCGAGAATACATATGATTACATTACTAAGGAAGAAAGACCGGAACTTAATGACGAATATGTAGCGAGACGTTGCATTTTCGGTAAAAAAGCAGGAAAAATATCCAGGTCGGATTTTAGTAGGATAAGATCTGCGTTGGATCATGCGATGATAAATAATACACATACAGCATTTGCCAGATTTATCACTGAAAATCTGACGAGACTCAATCACAAAGAACATTTTCTGAATGTGACACGTGCATATTCTAAACCTGATTCTGAAAAATTGATACAACCGAGATACTGGCAGTCGCCTGTAGTTCCAAAGGATAAACAAATATATTATAGCAAGAATGCGATTAAAAAATGGTGTGGTTACGAAGATGATATTCCGCCTCGTTCTGTGATAGTTCAGATGTGTCTATTGTGGGGGACTGATCATGAAGAGGCAGATCATATCCTTCGCAGTTCAGGATACGCGGCGCTTAGTCCTGTTGTACTTCGAGATCTTATCTATATGTATTATCTGGATCATCAGGATTTGCAAAAAAATGAGTTGATATGGGAAGTAAAAAAGCAGTTGGATCACTTCGATTTGACAAATAGAAATTATGATACAAATCCTTTTGATGTAGGGGGCAGCGTAAATGATCATATCTGTGAACTGAGCGAGCATATAGCGAAGGCTCATTATATTTATGAGAGGGCTAAGGAAGGACCATTGCAAAATGTAATTCGGGATATTTTGGGAGATACACCTGCCCTTTATTCTGAAATGGCATTTCCTCAGCTAGCATCTATAAACAGGTGTGCTTGCAATTCGCTTTCTTCATATCAAAAAAATATTTTTGATACTGACATAGCTATATATGCAGATGAAAAGGACACAAGAGGTAAATCAGACCGTATCCTTGTTGAGGGCGCATCTTCGAAATGGTATGAATTGAAGAAACGCGATGCTAATAATGTCAAAATTTCTGAAAAGCTGAGTATACTCAATACTATTCTTAAATTTAATAGTGTTTTTTGGGAAGAATGTTACCTTGATGGAAATATAAAACAATCGAGCGGAAAGCGATCTGAGGCAGGAAAAATTCTTTATGGTCGCGACAACGGAAAAGAAAATGTCGGAGTTTCAAAATTGGAATTGGTGCGGTATATGATAGCTGCAGGTCAGGAACAAAATCTGGGAAATTACCTGGTGAGTTCAGGATTTTGGAGAAAAAATCATATGCTGTCATTTATACAAGGCAATGATATAGCGCTTGATGAGATGGATGAATTGGATCTCTTAGACTATATTCTGATATATGCATGGGGATTTAGGGAAAATATCATTAAAAAGAACAGTAATGTGAATTCTTTGGATGAAAAGACTAGAAAAGTGCAGTTTCCGTTTATAAAGTTACTCATGGCAATTGCAAGAGATATCCAGATACTTATATGTTCAGCACATGAAAAAACAGTCGATGAGTCATCTCGAAATGCAGCAAAGAAGATAGATATATTGGGAAATTATATTCCTTTTCAGATTCATCTTCAGAGAACTAAAAAAGATGGTGGAAGAGTGGTAATGGATACATTGTGTGCTGATTGGATTGCGGATTATGAATGGTACATTGATCTTGAGAAAGGAACACTTGGATGAGCAGTGATGAAAGGATATTTAAAAAATTTTTGGAAAAAGGATCGATTTCTGAGCAGAAAAAGATGCTTTTAGAAGAAAAGAAATGTTCGGATAAACTAACTGCACTGCTTGGGAATTACTGCATACCGATAGACAATATTTCAGAGTCAGACGGAAAAATATATGCGGTCTATAAGCTTCCAAAAAATGTTAAACCTTTGTCCGAAATCATTAATGATGTATCCTTTTCTGATTGTACGATGAGAGTACGTTTGCTTCTCATAAAGAGAATTCTGGAACTCGTGTGTGCTTTTCACGAAAAAAAATGGTATTGTCTCAGTATTTCACCGGGAATGCTCATGGTTGAAGATTTTGATATACCGATGGGAAATGTCGGAAAAGTATTGATATATGATTTCAGAAATCCTGTTCCGTTCGAGTCAGTAAATGAAAGACATAATTTTAACGTTTCAAATAAATACACTTCACCGGAGCTGCTCATCCATTCAAGATATGACGAGTCGAAATCTGTGAGTGAAAAATCAGATTTGTATTCTGTTGCAAAAATTGCGGAAACAATAATAGGAGATTTTAACAGTATTATTGCAAATGGAAATTTGATACTACTTGCAATGCTTAGAGTTTTTATCAGTACAGGGAAAAGTCCGGAACCTGAGTATCGGTTTGAATCGTCGGAAAATATGCTTTCAGTATTTGAAAATTTGATCAAAGAAAATTGTTTTTTTGAAAAAAACGATTATACATCTATGTTTCATCAGGCGTATGACAATTTTTTTGAATGGCAGGAATGTTTGATATCACCGGATCACTTGGATAAAAATATGTTCGAGGCAGCTTTATCAAATCTTGAGGATCAGCTGCTTAGGGTTGATATTGATAAGTATAGAGCAGAGTACTTCTATAAGCTTCTCCGAGAGTTGTCTAATAAATATAAAAATACAATTACTGATGAACAAAAGGTAAGGTTGGCAATACTTGGAATCAGAGCGAAAAATAATCTGGGAAAAAGTTTTGATGCATTGGAAATATATGAGTCAGTACGTGATTTAGAAACTATGTTGGAGGAGATGGCAGAGCTTAGTCCTGTCATTGCTTCGACATATATGGATTGCTACCGATATGCAGATGCGCAGAAAGTGGCGGAAGAAAACATTATCAGGCTTCATAATAGTAATATTCGTATGGAGAAAAAAAGAATACTGCTTGGAAGGTCATATAGTTCAAAAGGGTGCAGCATGGGGTTTCAGCATATTCTTGGTGCGGATGAGTCATTTGAACAGGCTTTATATTTCTTTAACGAAAAGGACAATTTTTGGAAAGAAATATTTGAGAGCAGAAATTTAGAGGACAGCGATAGACTTATAAAGTCTTTACGAAGCAATACGCATATTACGCTGTTTCATTACATGCAATATGCATGTGAAACAAGGAGAAAGGAATTATATGGAGCACTTTCAGACAAATATTTTATAGGTAAAGAATGGACAGAAAGACTCAAAGCATATATAAGCAACAAGGATATATGGAAAAACTATTATGAGATATATATTCTGCTAAAGGGTATTTATTGCTTCTATCCAGAAGTCATGTGTTCGTCTGCGTTTTATGATGAAATCCAAAAAATGTACGATCTTGAATTTGAAAAGGAAAAAATGTTTTACCCATTGAGTCTGATAGAACTGTATCTTGCTCTGATAGAGATAAAAGTTAATGGGAGTCTGACGGAGAATGCCGAGAAGTTGTTTAAACAGGCATTGACACATGACAATGAAGTCAAAAAAGGAAATATGAATATTCAGACCGCCATTTGGTATCGAATATATGCACTGTATAACGATGTAAAAGATGAAACTGATAAGAATAAAAGGCTTTTAAAACGGCTTATGATTCTTTGCCGACGATTTGGTTGGGCGGATATGTATAGTGCTTTGGAGAAGGATGGGAAGTTAATTGATTTTTTGAGATTTGAGGTATGTTAAATGATAACACTTGCATTAGATGAAAATGGCAAATTTGAAGATGCTTTTTCTAAAAAAAATGAAAAACCGATAATGATTGCGGGGATAATCTATGATGACAAGGGGAAAGAGTATGATGCTGAGAATGAACGCTACAGGATATCCAGTTATCTGCGAGCAGTATGTGACAGTTTGGGTGCGAAATACCCTCAGGATCTACATTCAAATAGTAATGGAAATAAGGCGACTGTTGGGAAAGTAAAATGTAAAATTGGTGAAACACTAAAGGAATTCTTGAGAGAAGGAACCTATGAAAAAAAGGAATTGCCGACAAAGAACGGTTATTTAAATAAGAGATCTGGAAAATATGTAATGTTTGCAGAACTCAGGAGTAGTCAGGGAGTTAAAAAGCGTGTTAGTGGTTGGAATGACAATGATCTGACTCAGGATGAAAAGGTCAGCAATCTGTACCTTCATATGGCAGAAAATGCCGTTGTCAGAATGCTCTTCCATAATCCTATATATGAAGATGTAACAGATGTAAATCTCTATTTTCCCACGCGAAAAGTTGTTCTGAAAGATAGAGATAGAGAATACGATAAACAAGATTTCAAAATATATGGTGATAAGGACAAGTGCGAAGCAGAAAGCGGGAGATTGGTGCATTATGATATCGTGTCATCGGATTTTTACCGTACGATAATGGAGAACGAATGTACAAGAATTAATAAAAAGCAATTAAATGTTCATTATATGAACACAAGCCCAATTTCGTACTGGGAGAAAAATGAAAAATATAATACATTTTTATATTTGGCTGACATAGTTTGTTCTATGCTGGATTATTACAAAAAGGGTTCGAGTCCGGCAGAGTGGATGGATTCTTTTGCCGAATGGGGAAACAAATATTTTGGTGATGATCAGATAATCTTATTTGGGTATGATGATATAGATGACAAATACATGGAGGCTGTAGATGCAGTAGGACAGGGAGAGTATTTTCATGCGCTGGATATTATATATGATGCGGAATGTAGTGGAAGTGAATTTGAGAAGCACTACAAAGATTATTGGTTTCCAAAGCTTATAAAAAAGATACGAATAACAGCAACTGTGGATAATTTATGCAGATCGATCTCAGATCTGGAGAGTTTTACATATCGAAGTAATCTTGATCAGCAGAAACTTTTGTGGATTTTTGAGGAAATCAAAGCTATCGTCGATAAGGGAGATTTTGGAAAGAAATATCATACAGATCAGGTTATGTTTGATATGTGTAATGCCGGTATTGCTGTGTACAATCATATCGGAGATTTTGGGACTGCAAAGGAATACTATGATGAGTGCATGAAACACACTGGGGATGTGGATCTGGTAAAGATACTTCGTGCATCAAATAAAATGGTGGTCTTTCTTGACGATGCTTTTAGGTATGGTGACGCGACAGAACGTGCCAGGAAGAATGTTGAATACCAAAAAGCTTTGCACGATATAAAGAGTGAGATTTGTCCGGAAAAGAAAGATGAAGACTTGAACTATGCCATATCGCTCAGTCAATTTGGACAGGCGCTTGCGTGTGAAAAAAATTCTGATGCAGAGAGTGTTTTCCTAGAGTCGTTGCGGCATATGAGGAAAGGGACTGCCAATTATCAGATTACTCTTTCATATTTACTCCATTTTTATCTGGATATGGGAATGACAGATTCTTATCGAGAAAAAACAAAGGACTATTTTGGAAGTGAAAAACCAAAGGAACAGCTGAAAGAATTGCTGAAGTTATCGGGAAAGGATGATAGTATAGTTACTTTCAAATTTGCAATGTATGTCTATTTACGTGCACTTTGGGTATTACAGGAACCGCTTACTGATTTTATCAGAACAAGATTAGAGGACATACGTGAGACTCTTGTAAAGAAGAAAATGAGTGAACATATGGTTGGACATCCGTGGGAGTTGATTTATAAATATCTGGCATTTCTTTTTTATCGTGATGGAAATTGTGAAGCTGCTGAAAAATATATTCATAAAAGTGAAGAGTGCTTGGAAACACAAGGACTGACTATAGATGCGATTATTCATAATGGTAAGTATGAATATGCAGAATTGTCAGGTGACGAGGAGATGATGGCAAGAGAGAAAGCGTACTTTGATGAAAAAGGGATAGATAGAAAAAATGTTTGTACTTTTATGTATCATTGATGTTTAATAAGATTTGACCGAGGAGTGACAGGTAATCGCCGGTATATCTGGTATTACCTGTCATTTTTTGATGAAATAAGCTACTTTTTGCCTAAAAAACGAAACTGTTGGTGTTTTATGATGATTGTGTCAACAAAAGAGAGCAAAAGAAGAGGAGAAAAGTAATGTCAATGATTTCATGTCCGAATTGTGGTGGAGAGATATCTGAAAGGTCAAAGAAATGTGTTCATTGTGGATATGTGTTAGTCGAAGAAGCTAAAGTAGTGTGCACAGAATGTGGAACTGAGGTAGAGAGTGGCGCTGCTGTATGTCCGAAGTGCGGCTGTCCTGTAAATGATAGTGAGACGCCTCAGAAAGTTGAAGTGACTAGGGTAAATGTATCTTCCGTAATCAGCAAAAAAGTCGTTGTAAGCATACTGATCGCAGTGATTACAATTGCAGGTTTTTTCTATGGAGTGAAGTATTCGCAGGAAAAGAAAGCAATTGAAGAGTCAGTAAAGCAGAAGGAAGACTATCAAAGTACGCTAGAGCTTGCTTCGCTAATGATGCTTCAAGGAGCTTCGGATGCAGAAACTTGTGGGAATTTGGTTAGGAAAGTGTGGAGCAACTGCATTTATAAGGAGAGGGATGAAGAAACCGACAAGTATACGTGTGATAGCAGGGGTGCAGGATGGTTTTATGATGATTTTAATGATGCATTAATGGCTCTTTACAGTGACAGCAGTTTTGGCAAGAAGATAAATGAAATCAAAAACGGTCAGGAAACCGTTGCGGCGATGATGAAAGATCTGAAAAATCCGCCGGATGAGATGGCAGATGCCTATGAGGATATTCAAAATTTTTATGTGTCCTATCTAACGCTGACAGAAATGGTTGTGAATCCAACTGGAAGTTTGAGTTCTTTTTCATCTGATTTTTCCGATGCGGATACGGAGGTGTCCAATGCCTATAGCCGGATGAAGTTGTATTTAGATTAAACTATTGAGGAAAAAATGGAGGTGCTTTAATGCGGGGGAGAAACTGTGGAGGGTCATCAGGCGACGGACTGCTGGTACTTCTCGTACTGCTTGTCCTTTTTTATAAAATCATGCCATTCATAGGTTTATGGATTTTAATTTTTGGTGATGCTGAACGTAAAGATCTGGGTATGGGTATGATTATTGTCGGGATAGTTCTATATGTATTATTAGAGGTTTTTTAATGTGAGTTTCTGTGGTAAACTATAAAAGTACAAGCTTTTGCGCCGCACCGCATAAATAGCGGATTTATGACCATTATTTGGTGAAAAAAATGGTGTACACCTGTGTTTTTTTGTTTTGCGCCGCAAAATGCGCCACGGAACCGCATGCAGAGCACCCTGCAAGAGACAGGGTTATGAAAACAGCCCGACATAGAGGGCAATAGACACGGGGAGAAGTCATTTAATAAGGCCACTGTTAAAAGTTATGAAAACAGCCCGACATAGAGGGCAATAGACATAAAGACCAAAAACAGGTCATCTGCATACTGTGTTATGAAAACAGCCCGATATAGAGGGTGTGAGAGATATAGTTCTCGTCACAGTGCAGAAAATGACCTATTATGTGCCGAAAAACAAAATGAAAAAAGAATGGAAAGGCGTATTTAATGAAATGCTGATCTGTTGATTTGAATTAACAAAAAAAGGTCGCCCCACGGATGACAAAAACATCCGGGGGCGACCCTTTT >E-locus (SEQ ID NO: 76)TACTGTGTGCATAAGTCTTCCTTAGATCCATAGGTACAGCAGTTTTATTTATTAGCCTTAGAAAATGGAAAATAGAGCTTATAAATGATATGATATTTATGAATAAAATGATTGCATTCTCGTGCAAACTTTAAATATATTGATTATATCCTTTACATTGGTTGTTTTAATTACTATTATTAAGTAGGAATACGATATACCTCTAAATGAAAGAGGACTAAAACCCGCCAAAAGTATCAGAAAATGTTATTGCAGTAAGAGACTACCTCTATATGAAAGAGGACTAAAACTTTTAACAGTGGCCTTATTAAATGACTTCTGTAAGAGACTACCTCTATATGAAAGAGGACTAAAACGTCTAATGTGGATAAGTATAAAAACGCTTATCCATCATTTAGGTGTTTTATTTTTTTGTGATTATATGTACAATAGAAGAGAGAAAAAAATCATTGAGGTGAAAACTATGAGAATTACTAAAGTAGAGGTTGATAGAAAAAAAGTACTAATTTCTAGGGATAAAAACGGGGGCAAGTTAGTTTATGAAAATGAAATGCAAGATAATACAGAACAAATCATGCATCACAAAAAAAGTTCTTTTTACAAAAGTGTGGTAAACAAAACTATTTGTCGTCCTGAACAAAAACAAATGAAAAAATTAGTTCATGGATTATTACAAGAAAATAGTCAAGAAAAAATAAAAGTTTCAGATGTCACTAAACTTAATATCTCAAATTTCTTAAATCATCGTTTCAAAAAAAGTTTATATTATTTTCCTGAAAATAGTCCTGACAAAAGCGAAGAATACAGAATAGAAATAAATCTCTCCCAATTGTTAGAAGATAGCTTAAAAAAACAGCAAGGGACATTTATATGTTGGGAATCTTTTAGCAAAGACATGGAATTATACATTAATTGGGCGGAAAATTATATTTCATCAAAAACGAAGCTAATAAAAAAATCCATTCGAAACAATAGAATTCAATCTACTGAATCAAGAAGTGGACAACTAATGGATAGATATATGAAAGACATTTTAAATAAAAACAAACCTTTCGATATCCAATCAGTTAGCGAAAAGTACCAACTTGAAAAATTGACTAGTGCTTTAAAAGCTACTTTTAAAGAAGCGAAGAAAAACGACAAAGAGATTAACTATAAGCTTAAGTCCACTCTCCAAAACCATGAAAGACAAATAATAGAAGAATTGAAGGAAAATTCCGAACTGAACCAATTTAATATAGAAATAAGAAAACATCTTGAAACTTATTTTCCTATTAAGAAAACAAACAGAAAAGTTGGAGATATAAGGAATTTAGAAATAGGAGAAATCCAAAAAATAGTAAATCATCGGTTGAAAAATAAAATAGTTCAACGCATTCTCCAAGAAGGGAAATTAGCTTCTTATGAGATTGAATCAACAGTTAACTCTAATTCCTTACAAAAAATTAAAATTGAAGAAGCATTTGCCTTAAAGTTTATCAATGCTTGTTTATTTGCTTCTAACAATTTAAGGAATATGGTATATCCTGTTTGCAAAAAGGATATATTAATGATAGGTGAATTTAAAAATAGTTTTAAAGAAATAAAACACAAAAAATTCATTCGTCAATGGTCGCAATTCTTCTCTCAAGAAATAACTGTTGATGACATTGAATTAGCTTCATGGGGGCTGAGAGGAGCCATTGCACCAATAAGAAATGAAATAATTCATTTAAAGAAGCATAGCTGGAAAAAATTTTTTAATAACCCTACTTTCAAAGTGAAAAAAAGTAAAATAATAAATGGGAAAACGAAAGATGTTACATCTGAATTCCTTTATAAAGAAACTTTATTTAAGGATTATTTCTATAGTGAGTTAGATTCTGTTCCAGAATTGATTATTAATAAAATGGAAAGTAGCAAAATTTTAGATTATTATTCCAGTGACCAGCTTAACCAAGTTTTTACAATTCCGAATTTCGAATTATCTTTACTGACTTCGGCCGTTCCCTTTGCACCTAGCTTTAAACGAGTTTATTTGAAAGGCTTTGATTATCAGAATCAAGATGAAGCACAACCGGATTATAATCTTAAATTAAATATCTATAACGAAAAAGCCTTTAATTCGGAGGCATTTCAGGCGCAATATTCATTATTTAAAATGGTTTATTATCAAGTCTTTTTACCGCAATTCACTACAAATAACGATTTATTTAAGTCAAGTGTGGATTTTATTTTAACATTAAACAAAGAACGGAAAGGTTACGCCAAAGCATTTCAAGATATTCGAAAGATGAATAAAGATGAAAAGCCCTCAGAATATATGAGTTACATTCAGAGTCAATTAATGCTCTATCAAAAAAAGCAAGAAGAAAAAGAGAAAATTAATCATTTTGAAAAATTTATAAATCAAGTGTTTATTAAAGGTTTCAATTCTTTTATAGAAAAGAATAGATTAACCTATATTTGCCATCCAACCAAAAACACAGTGCCAGAAAATGATAATATAGAAATACCTTTCCACACGGATATGGATGATTCCAATATTGCATTTTGGCTTATGTGTAAATTATTAGATGCTAAACAACTTAGCGAATTACGTAATGAAATGATAAAATTCAGTTGTTCCTTACAATCAACTGAAGAAATAAGCACATTTACCAAGGCGCGAGAAGTGATTGGTTTAGCTCTTTTAAATGGCGAAAAAGGATGTAATGATTGGAAAGAACTTTTTGATGATAAAGAAGCTTGGAAAAAGAACATGTCCTTATATGTTTCCGAGGAATTGCTTCAATCATTGCCGTACACACAAGAAGATGGTCAAACACCTGTAATTAATCGAAGTATCGATTTAGTAAAAAAATACGGTACAGAAACAATACTAGAGAAATTATTTTCCTCCTCAGATGATTATAAAGTTTCAGCTAAAGATATCGCAAAATTACATGAATATGATGTAACGGAGAAAATAGCACAGCAAGAGAGTCTACATAAGCAATGGATAGAAAAGCCCGGTTTAGCCCGTGACTCAGCATGGACAAAAAAATACCAAAATGTGATTAATGATATTAGTAATTACCAATGGGCTAAGACAAAGGTCGAATTAACACAAGTAAGGCATCTTCATCAATTAACTATTGATTTGCTTTCAAGGTTAGCAGGATATATGTCTATCGCTGACCGTGATTTCCAGTTTTCTAGTAATTATATTTTAGAAAGAGAGAACTCTGAGTATAGAGTTACAAGTTGGATATTATTAAGTGAAAATAAAAATAAAAATAAATATAACGACTACGAATTGTATAATCTAAAAAATGCCTCTATAAAAGTATCATCAAAAAATGATCCCCAGTTAAAAGTTGATCTTAAGCAATTACGATTAACCTTAGAGTACTTAGAACTTTTTGATAACCGATTGAAAGAAAAACGAAATAACATTTCACATTTTAATTACCTTAACGGACAGTTAGGGAACTCTATTTTAGAATTATTTGACGATGCTCGAGATGTACTTTCCTATGATCGTAAACTAAAGAATGCGGTGTCTAAATCTTTGAAAGAAATTTTAAGCTCTCATGGAATGGAAGTGACATTTAAACCACTATATCAAACCAATCATCATTTAAAAATTGATAAACTCCAACCTAAAAAAATACACCACTTAGGTGAAAAAAGTACTGTTTCTTCAAATCAAGTTTCTAATGAATACTGTCAACTAGTAAGAACGCTATTAACGATGAAGTAATTCTTTTAAAGCACATTAATTACCTCTAAATGAAAAGAGGACTAAAACTGAAAGAGGACTAAAACACCAGATGTGGATAACTATATTAGTGGCTATTAAAAATTCGTCGATATTAGAGAGGAAACTTTAGATGAAGATGAAATGGAAATTAAAAGAAAATGACGTTCGCAAAGGGGTGGTGGTCATTGAGTAAAATTGACATCGGAGAAGTAACCCACTTTTTACAAGGTCTAAAGAAAAGTAACGAAAACGCCCGAAAAATGATAGAAGACATTCAATCGGCTGTCAAAGCCTACGCTGATGATACAACTTTAAAAGGAAAAGCAGTGGATTCTTCACAAAGATACTTTGATGAAACGTATACTGTTATTTGTAAAAGTATCATAGAAGCATTAGATGAAAGCGAAGAGAGATTACAACAATATATTCATGATTTTGGAGATCAAGTGGATTCTTCACCTAACGCACGAATTGATGCGGAATTACTACAAGAAGCAATGAGTAGGTTAGCTGACATAAAGCGGAAGCAAGAAGCACTTATGCAATCCTTATCTTCTTCTACAGCAACGCTTTACGAAGGCAAGCAACAAGCGTTACACACTCAATTCACGGATGCGCTGGAGCAAGAAAAAATATTGGAACGCTATATTACTTTTGAACAAACTCACGGGAATTTTTTTGACTCATTTGGAGAACTTGTCTATCGAACGGGACAAGCAGTGCGTGAATTAGCTAATAACGTCACATTCGAGAGCCAAACAGGAAGCTATCATTTTGATAAAATAGATGCTTCTAGATTCCAAACTTTGCAAGAAATGTTGCCAAAGGCAAAGAAAAAAGCATTTAATTTTAATGACTACCAAATAACATGGAATGGCACCACGCACCTTTTATGGAAAAATGGTAAAGTGGATGCAGAAGCAACCAAAGCTTATAACGAGGCGAAACTGAATGGAAAGCTACCAAAGGAAGGTAATGTAGCAACACAAGATGCAGAACTATTAAAAGGCATTTTGGCTTCACTGAAAAACAAGAAAGATCCTATCACTGGAGCAGATATAAGCAGTGTGCATGTATTATCTATCCTTAGCGGGCTCGCATTCTCCTATACAGCTGGGAATTATAAGGGAAGAAAACTTACTGTTCCAAAAAGTTTCTTAGACAAATTAAAGAAAAACCGAAAATCTAAAGTACCTAAACTATCTAGTTTATCAGAAAAACAACAACTAAAACTCGCAAATAAATACAAGAAAAAATCACCTATTCCAATTCCAGATGATGCTAAAATCAAAGCTCAGACGAAAAAGGCTGGTTATGAACAAATATCTTATAAATGGAAAGAGAATGGGATAACCTTTGAAGTTAGATGGCATACTAGGACACCAGGTGCACCAAAGGAACAAGGAAATACGTTTGTTATAGAAAGAAAAATTCAGGGTACAGCAGAAGGGAAAACAAAAGTTCAACAAATATTGGTTGGAGATAATAAGTGGGTGAGTAAAAGTGAGTGGCAAAAGGCTATAACTGATAAGAAAAATGGTGTAAGTACCTCGGAGCAAAATAAAATGTTGTCTGATGGACATTGGAAAGAATAGAAAGGAGCAAAATGATGGAAGATTATTATAAAGGTTTTGAGGGATATCCAGAGATAGATTTTTATACGTATATAGATGATATGAAATTGGGTATAGCAATGTGGGAAGGATACTTTGACAACATTATGAAAGAAATTAATCCAAGTAACGGAAGATGGACTTCATTAGCGTATTATTATCATTTAGATGAGGGGTGGTATGATGAAAGTCCTTGGGAAATACCAAGTAATACAGAAGCATTAGAATTATTGGAAACAATCCATATATCTAATCTAGATACTATCACACAAGAGATATTACTTAAATTAATAAATTTATTAAAGAAGAATATAAATAGACAAGTTTATATTGAATACTCATAAAAAAGATGATTATGATATATTATAGAACAAACGAACAAGCCCCAAATACGAGGTTTGTTCGTTTGTTTTCAATATAATTATTTGCCACCAAGTGAGATATTACGGTTTTAAATAGCTTATTTGACGATACCAAACCCTGATAAGAGAAAGAAGAAAGAGAAAGCTGGTGTAGTTGTTTTAAGTGAACTAGATAAAAAATTAATAGCAAAACTTGAAAAAGATGGTGTGAAAATATCAAAAGAAGATGTTATAGGAATAAAATAATTGCCAGATGATGAGAAATCGTTTGGCTGGAAAAAGGAAATCCATCCGCTGGATTTGAGCATATTCTTATTGAACATGGTGAACAATTTGCTAAATAGGGAATTTCAAAAGCTGAGTTACCTGATTTTTTGATGACTGCTTTAGAAAAGGAAA >F-locus (SEQ ID NO: 77)ATTCTTTAAAAATATCTAATAATTTATTTACTATATACTCTAATACATCTTTTAACCTATCTAAAACATCATCACCTACAACATCCCAAAAATCATCTAAAAAGTTAAAAAAATCCATCTTTATCAACTCCTATATCTATTTTTTATTGTGTAATTCCTGAGTTACAAAACCATTATAACACGTATTACACACGTAGTCAATACTTCAAAAAAATTTTTTGTATATTTTTTTGAATAAGTAAATAAAAAGAGCTGTGTAGCTCTTTATTAAAATCAATATTTTTATTTTGTTAACAAACTTAGACAACATTAAATTTAGAAACCTATATATATTTCAGTACTTTTCATTTTTAGGTAGTCTAAATCAGAAATGGTTTTGTCTAAATGATGTATGTAAGTTTTAGTCCCCTTCGTTTTTAGGGTAGTCTAAATCAGAAGTCATTTAATAAGGCCACTGTTAAAAGTTTTAGTCCCCTTCGTTTTTAGGGTAGTCTAAATCCCATCCAAATTATGGGATAATATGTTACTTTTTATTTTAATATTTGATTATTTATTGTTTTTTTACTGATTTAGATTACCCCTTTAATTTATTTTACCATATTTTTCTCATAATGCAAACTAATATTCCAAAATTTTTGTTTCTTTTCTTATGATCTTTTCTCCGATAGTTATTTCTCCAGATAAGATTTTCATTTTTTTGAATTGATCTTCTGTTAGAATTAATGTTCTTACTGATGAATTTTCTGGAACTATCATTGACAACTGATTTTCATAGGAAATTATTTTTTCTTTTGTGCTAGAACTTACAATGTATACTGATTTTTGTACCTGATAATATCCTTTTCTTATAATTTCTTTTCTAAATTTTGCATATTCTTTTTTTTCTTTTCCTGTTTGCATTGGAAAATCATACATTAGAATCCCTACATAATTAGTACTCATAATCCTCTATCCTTAACTCAGGAATTTCTACTTCTGACATTTCTCCTGTAAAATAATTTCTAATATTATCTAAAAAATAATCAATCACTTGAGCCAATTCATATTTTTTATTTTTCCAATAAACTTTTTGTGTTAATACCAATAACAATTTTTGTCTTAATGATTTATTCAAACTTACTTCTTCCTGTTGATTAAAATATACGATATAATCTACCATTGGACGAAATATTTCAATAATATCATCTGCAAAATTATAATTATTAAATTGTGAACTGTGATGTATTCCCAAACTTGGATGAAATCCTTTAGCCACAATTTTTGAAGAGATTAAGCTTCTCAAAACCATATACCCATAATTTAATGCCGAATTTGTCCCGTCTTCACCAAATCTCTTAAATTTTTTCCCAAAAAGTTCACCAAAATACATTCTTGCAGCAATTGCTTCCTGATGTTCCGCTTCTTTTCCTTTTAATCTAATATTATTTTCATATGCTTCCAACTTATATGATACTTCCTGAGATTTTTTCAAAAACTGCAATAAATTTCTTTGATTTTCTATTTTTCTCATTACAATTTTTCTCCAGATTTCTTCTTTTTTATCGTCAATCCAGCTCACTTGCTCATTAATTCTTGTTGTTACTTGAAAATGATTATACAGTCCTAATGAATGTAAAACTGGCTGATGTTTTTCATTACAAATTATCAGTGGAATATTATGTTCTGATAATCTTAACTGTAATATTCCGCTAATTTTACATCTGCAATTTTCAACTACAATTGCCATGATATCATTTAAAGATACTTTATCAGCCTTATTTTCATCATCTTCATTTATCATCACAAGCTGGTTATTTAAAACTGATAATTCATTGACTCTTGTTACATGGATAATATTAGACATTTTTATTACTCCTTTACTCTAAAGCTTTATATTCAAACATAACTTTCACAAGTTCACACAATTCTTCTGAATTTCTATCAGTCATTAATTTTTTCTTTTTTAAATTTTTCAAATGTACAATTTTTTCCGATTCTAAAGTCTGAATTTCTATTTTCTTATCTGCTCCTATTTTAAATGTTGCTACAAAACCATATTCCTTTAATATATCCACTATTGATTTCATAATTGCATTTTTAAGTTTTCTATCATAAGAAAGTAATTTTCTTAAATTTTCCAGCACTTCTAAAAGTGAAATTTCAGCATGCGGAATATAGTTAAAATGTGCAATATAGTTTCGTATATACAAATCTTTTTTCTCTTGTTTTAATTTTTTTACTTTTTTATCAGAATAGATGCTTCTTTTTTCTACATTATCTTTGTATAATTCTTTATAAAAATTTATATATTTTTCAACAATTTGCCCACTTTTATATTTTACATTTTTACTGTTATCAAAATTAAATATTTCTTCAATATAATGATTTTCAGGAAATTCACCTTTCAATCTAAATCTTAAGTCCCTTTCCCAGATCGAAGTATATCCCACAAGTCTGTGGAGTATTTTTAATAACAAGCCTTGCAACAAGTTTAATTCATTAAATTCCACTTTATTTTTCAAATGAGTATATTTTTGTATATTTCCAATTGCTTTTTCATATTCTTTATAATCTTCATCATTAAATTTTTCATCTTTTTTAGGTCTTGCATATTTTCTATGTAAATTTTGCTGCATTGTATAATTTTTTTCTATTTCATTTTTTTTATTGCTGTATTCTTTCAATTCTTTTAAACTTATTTTATACTTCGCTTTATCAGCTATTTTTTCAAGTAAATTTAACATCCCATATTTTTTTATATTATAAAAAGCTCTATGCTTTATAATATTTTCTCCATCAAAATATATTTTATTTGTGTCAAATTTCTTCAATTCTTTCCTATCTTTTATTTTATTTTCATTAAAATCTAAAAATTTTCCAATTTCATTCGCTTCTAATTCAAAATCTTCTGTTACTCTATTATTATCTAAATTTAAAAGATTTATAAGTTCAAGTTCATCTGAAAAAGTTTCTTCTTTATTTGCACTCTGATATTTTTCAAGACTTCCCTTCAAATTAGTCAATTCTTTATGATTAAGCAATTTTAAAATTAAATAAAACATATTCAAATTTTCAGTGTATTTTAATATCTTTCCTAATTTTATCTCTCTTACAAATTCATTTATTTCATGTGGAATTTCTTTATTCCTATTATGTTTTTCATAATTTTTTAAAATTTTATCATATTTTTCTTTATTATCTTTTTTTATTTTTATTTTAGAAAATATATCATTATTATCATTGTTATTATTACTTTCTATATATTTTAAATTATTTTTATTCAAATAATCTATAAAACCTTTTAAAAATATTTGTTGTATAAAATCAATGTATGTATTTTTTTCTTCTTTATCTTGATTATTAATCATCTCCCTACTTTGTATAATAGCAAGATATTCTACTGGTACAGTTTTTTCTATATTTTCAAATTTTTGATATTTATAATGTCCTGTTTTTTGATTTCTTTGTTTATTTATTTTTATTACTTCATTAGTTATTTTAAAAAAAACTTTACTATTTTTAACAAATTTATTAAGAAATTCACCATAATAAATATTTTTCAAAAGATATATTTGAGCATCTTTTTCTTCTTTATCCTTAGGAACACTCCAAAAAAATTTTAAAGTATTTCTTAAATCTTCTATTTTATTATATAATTTCGTAAAAGAAGGAACAAAAGGAATATTCTTATTTACAAAATTAAATTTTGTATTTTTTAAATATTTAATTATCACATCCTTTTCATAATAATTAAATACATTTGCACTATTTAACTGCTTAAATATCTTCAATTTCAATTTTTTCTCATTTATTTCATTTTGAAACATTTTTTTTGAAATTTCAGAAGGAGCTATATTTTTAAATGCAAATATATCTTTCCCTTCTAATTCCAAATTAAAATGCACAATCCCATGTCTAATACTGCTAATAGCTTCATCAATATTTGCAAAAAAATCTTCTATCTCATTTTTATTATCCATATTAAAATCATAACTATAGAACATTTTTAAATTTTCTTTTACTTCATTTTGCTTGTTTTCATTATATATTTTATCAACTTCTCCAGAAACATATTTTTCTTCGCCCTTATTATTTTTTACAGTTTTTCCTCTCATTCTACCTGTAATATCATTCTCATTTTCAGTTTCAAGAATATTTCTCAATGAAAAATATGCAACCGAAGAAACTCCAATTATATTTCGTAAAAATGCTTCATTTTGTCTATTCCTAGCAATAAAATCACTTGTTGCAATCTCTCCAACTTGTAAATAATAATTGTATTTCCCACAATTTCTTACATAAGTATCCAATTTATTTAGTAATTTGTTTTCAATTAATTTTTTTAAATTTTGATATTCAAATATTCTCTTAATTTTATCGTTACTTATGTTACTCAGTCTTTTATACACATAATTTTTCAAAAGCTGACTCATTTCAATTTCCACAAAATGACAAAAAGCATATTTTATATTTTTATCATTAAGTTCTTCTTTATCCAAATAATATTTATAAAACACTTGTGATTTTTTTAATTCACTCATATCCGGAATTTTTTCAATTAATTCTTTTATATTATTTACATTTTGTATTTCTTCGTAAATAATTTTAGCAAAATTTTCTTTATCATTTTTTCTTCCAATTATTTTGTGATAGTATTCTCTTATTTTATATTTTTCATGTTTTTTTGAATTTTCTATTAAAAAAAATAACTTCTCAATATCTTCTTTTTTATACAATTTATCAAATGCTTCCTGTACATTATTTATATAATCATTACGCTTTGCTGATTCTCTATAATAATCATAAATAATATTTCTTTTGCTCTTCCCTCCAACTTTTTCAACATTATTTTCATTAATTTTCTGATAATTAGCCTTATTTTCTTCAAATGAATATTTTAAAGAATTTATCTTATTCAATTTTGCCTCAACATCTTTTCTAAATATTTCTAATTCTTCAGAGTTCACATCTTCATTTAACAATATTTTCTTTAAAACTGAAAAACTATTTTTATTTTTTAAATCATATTCTGAAATATCTTCTTCAGAATAATTTTTATCCTGTACTGCATTTTTCTCTTTCCTATTCTTTAAATACAGAACACTATCTTTTAGATGCAATACTTTATTTGAAAAAAACTTTTTTAAATTTTCTCTTCTTATTCTATTTTCTTCTTCACTTGCATTATCAGGATTTTTTATATATATATCCAGTCTTATACTTAAAAGCTCTGACAATCTCTCACTAGTCCTATTTTCTTCGCTCGTACTTTTTACTAATTTTCCCTCTTCAATATATTTTTTATGCGAAATTCCATCAACTTTTGTAACTTTCATATATAAAAACCTCCTAATATCTATATTTTTTACTCAATACCTAATTCTTTTTTCAATGCTTTTTGTAAAATTTGTGAAAAATTCAGATTTTTTTCCTGTGCCAATATATCTAACCAAACAGGAATTGTTAAAGTTTTCTTTTTAAGTGCATTTGTAACTTTTGCCACTTCATACACTGGATCAACAGATAAAATATACAAATACTGATTTTCTTTCAGTTTCACATCCTCCACTTTTGAAGGCTCAGGAAATTTTTTTCTTACATCCAAAAAATCAGCCAAATGCAGACCCAATGTCTCTCTCAAATTGGAAACAGCCTCCTCCATGCTATCTCCAAATGTAGCATAATAATTTATCTCTCCATCTTCAAACTTATCAAAATCAACAATACAACCATAATAAGTCCCATCTTCCTTAGTTACCACTGCTGGATAAAATACATCCATTTTAATTATCTCCAATCTATACCACGTGTTAAATACGTGTTTAAAAATATTTATAAAATTTTTTAGCATCTCTGCTAAAATAAAACAATTATTTCAAATTTTTCTATTCCTTAATCACTCATTGTTAGTGATTCTTTTTTTACTTGGACAATTTTTCATTTAATTTCTTCAATTTTTTTAAAATCACATTTTTTTAATATTCCTTATTTAATTGCAAATTTTCATTACTTTTGGGGTGCTCTAAATCCCATCCAAATTATGGGATAATAATTTTTAGTGAAAGCAAGAAGGGACTAGAATTTAATCCCAACTTGTTTTTCAATACTTCTTAATGTTCCTACAGGTATATCTTTTGAATATGGTACTGTGACCACACCTTCCACACCTGGGATCATCCATTGATAATGACTACCTCTTATACGCACAACTTTTCCGCCTAATTTTCTAAATCTTTTTTCGAT >G-locus (SEQ ID NO: 78)CTTTCTATCTTTTTCAAATAAAATTAGGCTCTAGTTAGCCTAATCGCATAATTATTTATTATAGTATAATTCTTATTTTTTTTCAACCTAAAAATTTAAAACATCTCCAAAAATTTTCGTTTCAGAACAACCAAGCAACCATATTCAAAAAACAATAAAAAATGAGCAAGAATTGAAATTTTATTCTCACTCAGAAGTTATTTTTATTAAATATCACTTTTCGATATTGGGGTGGTCTATATCAATTTAAAAGACAGAATAGATAATTCTTTAGAGTTTTAGTCCCCTTCGATATTGGGGTGGTCTATATCAGAAGTCATTTAATAAGGCCACTGTTAAAAGTTTTAGTCCCCTTCGATATTGGGGTGGTCTATATCCCATCCTAATTTCTTGCTGATGAGATATTTATTTCTAATTTTTCTATTTTGTCTTTATTTTCAATACTTTCAATCCTATTTTTCTCTTTATTAATAATATAGAACCACCCTATACTATTATACCATATTTTTTGATTTTTCAAAATTCCAATATTTTGTTTTGTGAAATTTTTTCTCCCATTGTCACTTCTCCTGCAAGTACCTTCATTTTTTGAAACTGATCTTCTGTCAGGATAATGGAACGGATTGATGAATTTTCTGGAGCGAGCATTGATAACTGTTTTTCTGCCAGTTCGATTTTTTCTTTTGTTTTCGACCTCATTATATATACCGATTTTTGAAGCTGATAATATCCCTTTTCTATCAATTTTTTCCTAAAAGTCCTATATTCAAATCTCTCAACATCTGTCTGCATAGGAAAATCATACATAAGCAGACCAAAATACTCAATACTCATAGTCCATCACGCTCAATGTCGGAATTATCACTTCTTCATCTTTTACAAAATAATTTCGTATACTATCCAAATAATAGTCTACCGCTTGGAAAAAATCATATTTCTTATTGTTAAATAATACCTTCTGCTGTGCTACAAGAAGTATTTTTTGCCTTATTTCCTTACTTAATTTCACTTCATTCAAAATATCCTTGTACATATAAACAAGATAATCCACCATAGGACGAAAAACCTCTATTATATCATCAGAAAAATTATAGGCATTAAACTGTGACTTATGATGTAATCCTAAACTTGGATGAAATCCTTTTGCTACAATCTTTGATGATATTATAGCTCTTAAAATCATATATCCATAATTAAGTGCAGAATTCACTCCATCTTCATCAAATCTTTTAAAACTATTACTATACAATTCCTGAAAATATATCCTTGAAGCTATTGCTTCCTGATGTTCTGCACTCGCATCATCTTTTTTCAAGTTTTCCTTATATGTTTTCAGTCTTTCAATGGAAATATCACTTTTTTCAAGATACTCTAACAATGCTCTTTGATTTTCAATCTTATTCTCCACTATCCTGCTCCACAATTTTTCCTTTTTCTCTTTTTCCCACTCAATCTGCTCATTTATTCGTAAAGTCACTTGAAAATGATTAAATAATCCCAGCGAATGAATTTCAGGCTGATGTTTCTCGTTGCAAATAATAATCGGAATGTTATTTTCCACCAGCCTCAACTGCAAAATCGCACTAATCTTACAATAGCAGTTTTCAATAACTATCGCAGATATATCATTCAAAGAAATCTTATTTTTCTCATCATTATTGTCTTCATCAACCATTATAAGCTGATTATTCGATATTGACAAATCATCAGCCCTTGTTATGTGAATTATATTGGGCATTTTAATCATACTCCTTATAAATTTCATTCTTATAACGTATCATTCGTATTTTCTATTTTTGTTAAAAGTTCTATTATCAAGTTTTTAATATAATCAGAATTATAACTTTCTAATTCTAAAACAGAAACTTTTTTAGGTTTCATTAATCTTTCAAGTATATCATTATTACCGATAAGTTTAAATTTTTTCTTTAATTCATCATAATCTAAATTCACATCTTTTTTAAATACTTCAAATACACTTGCATAAGTTGAATTATTATAACGTGTACTATATGATAATAAATTAGAAACTCTATCAATTTGTTCTGCAATACTGTAATCAGCAAACGGATTTCTTACAATATAGAAATGTGAAATATAGTTTCTAATACTTTCATTTTCCGGCTTATTAATTTCAGAATTTTCAGACAAATCAATTCCAAATCCATAACATATTTTCTCAAATTTTTTATAAGATTCTTCATCAAAAAATTTATAGTATGCTGTTGTTGTATAAAAGCCATCAGATCCATTACGCTTAGGATAAGCTCTACTTATTCCAGTATTGTAGCCACTTAACTTAATAATTCCTAATTCTCTTAGCCCATTTACAATATAGTGCATATCTCTTTCAAATCTAGCCATTTGAATAGCAAGTTTCCAATTTATATCTATCAAATAACTTTCTATTTTATTCAAATAATTAAATTCTACCAAATCTCTAATTTTTTTGTATTCAGAAACTCTATTATAATCTTTTTCAAATGATTTATAGTTTTTATTTTGTATATTTTTTGCAAAAAAGTCATCATTTTCTTTCAATTTTTTTATATACTTCTCTTTGTATTCTTTAGAATATCCATTTAGTTTATCATTTAGATTTTTCAATATTGCATCAATTTCAGATATTTTATTTTTTCTAATATTTTTACCATCAATATTAAATAAAAATTTTGCATCAGCCATTTTAATATCATTTGAAATTAATCCATAAATTTTATCAAAATTTGGATTTCCAATATTTAAAAATAAATTCTTTTTATAAATATATAATTCATTCTTACGTTCTTTAGGATAATATATTTCTTGAAATTTATTTTCATTCTCTGATTCCATATCTTCTATTAAATTATCTATTTCTTTTTTGTATTTTTTTAAAAAATCAGAATTAAATATTATTCTACACAATATTTTACTCTTTATTTCCTGATCTTTATCTTTTATATACTGATCAACCTTTTTTTTCAAATCCTTTTTATTTATGTTTGATAACTTTCTTTGTTCATCTTGTAATATATTCGATTTTTTATCTATCTCAAATTTAGTTTCATCATCAAAAATTACAATTTTTTCTAATTTTTTCTCTAAAACATCACAACCATTAATATCATCTTTAAATTCAGTTAATATATTATTTTTTATATCCTCATAATAATTATTAAAAATTTCTTTTTTAGTTTGTATTTTAAAATCATCAAAGTCTTTTTCTATCTCTTTCATTTTTTGAATAAATTCTTCTAAATTAAGATTCCAATTTTCAGTTATACATTCATTTCTCAAAGTATTTAATTGCATTATTTCATCTAAAATATCTATAATATTTTGATATTCTGAAGTATTTAACCAAACTGATGTTGCAAAAAATCTATTTCTAATTTTATTTATAACCGCATTACTATTTAACAGTGCAAATATTGAAATTATATATTCAAAATCATCATTTATTACTATAGTTTTATCACTAGTCTTTACAGTTATTCTTTCGTAAGTTTTATTATCATTAATGTCTTTTATTTGTTTCTTAATTTCTTGAATATTCATTTTAAAATCTGAAAAATCAAAAAGTTCCTCATAATTTTTTCTCAAATATCCAATATAACATTCTATTACTTTTTTCTGATATTTTTTAATAGCTTTATTATTACCTTTTGAAGCAGAAATCTGAGCATTTTTATAATAATTTTCTATAATATTTTCATCTATTTCATCAATGTTTCCTAAAGTTTTCTTTAATTCTTGTAAAAATATATTCTTACTTTCATTTTCTTCTAAATCATCTTCTAAAATTAATTTCTTATACAATTCTTTATTCACATATATTAAAGCATTTAATACTATTTTTTCTGTTTCTATAGTATCAAATGGTTCATTCTTAGGATTATTCCTATATAAATTTAATATTTCAGGAAGTACTTTAGAAAAGGATGGTAAATATTTAATATCATTATTATTTTCTTCTGAAATTTTAATATCATTTATTTTAGTAATTATATTTTTTTTATCTTTAAATACTACATCTAAATTTAATGCTTTTGACACTTCTTCATCTGATATTTTTAAATTTTGAATTATATTTATGACTTTATTATAGTCATCTTGCGTTCCTTGTAAATCTCTTTCCTTGCTAATCGCATGTAATATCCTGTTTCTTTCATTTGTTCCTATCTTTGTAAATTTCCTAATAAAATTATTTGTAATGTTATTTTTATTATCTATAAAATCTAAGTCTCTTATTATTTTTATTTTTGAATTTAAAATTTTTTTATCAAGTACGTAATTTTTTTCTCGATCTCCTCCAAAGAAATCTATATTTTCATCATTATTTATATTTTCTCTAGAAAAAATCTTATTTAATTCCATATTGGTAGAAGCAAAAAAAGTAATCAATTCTAAATCCAATTCCTCTTTAGCGTGAAGTCTAGAAAAATCATCAGTATTTACTGTTGTCATATCTATATCATTATGTCTTAATTTCCCTAAATACATAATATGCTCTAACGTATATTGCTTAACTCTTTTTAAAATTTTTTCAGATAATATACTTTCATTTAAAATTTTTTCTATTTCTATTTTTTCCATTTTCTTTAATCTGACTTTTTGTTCATTTACCAATATTTTTTCAATTCTTCCTTTCAAATATCGATATATGATTTTATATAGTTCTTTTTCTTCATCAGATTTCTTTGAAAATTTTTTCGAATCAAAATTAACTTTATAATGTTTTTTAAATATTCCAAAAATTTCTGTATCACAATTTCCTTTTTTTAGTTCTTTTTCTAATTTTTTTATTAATTCATCTATTTTAAATTCTGCTAAAATTTTTTCTATTTTTTCTTTTATACTATTATTTTTTATATTTTCTACAAAAAATTTTACAATTTTATCTTTTTTATTTTCTCTTTCTATTTTAAATTTTTCGTGCTTATCTAATAGTACATAAGATTTTATATATGTTCTATTTCTTCTCTTTTCAAGAAATTCATTATTAACTTTTTTTACTTTTTCAATTCTTTTAGTAATATTCCAAAATTCTAACTCTTTTATAACAAAATCAGCTATATCTTCTACTGTTAAATCTACATTTATATTTAAAATTTTTTCAACAAGCATTTTTTTATTTTTAGATTTCTTTTTATCACCACCAACATTAAGATAAAATTTTACAAAACCCAGAATTTCTAAATTACTTTTTATTTTTTCTCTTATTTCCATAAAATTAGTCAAAATAACATCTATTTTATCATCTTTCAATAATTTTTCTCTTAAATGTTCTTCATAATATCGATTTTCAAATACTTTTTCTGTTTCATTTTCAATTATTTTTTCTATAATCTTATATAAACTCATGTTAATATTTTTAAAAATTTCGTAAATTGATTTTTTTGTTTCTAATTCATCATTTTCTATTATTCTTAATATTATTGAACAATCATTTAGTGTTTTATTAGTATACTCATCTCTGATATCTATCTCTATTTCTTCTTCATTCTCTTGTCTCTTTATTTCTATTTTTTTATCATCTTTAGTTATTCCTTGCCTAATTGCTTCATCTATTATTTTCTTTTTTGTAATCCCCAATGCTTTCAATTTCTCAGATTTTCCATATGCTTCTATATATAATACAACTTCTTCTGTTTCCAAAAAATCATCATTATTTTCTATTCTTATGATTCCTTCTTTACCTTTCAACTTAAATAGAATATTTCCTGCATGAAATTTTCTTGTAAATTCTTTAAGAATATTATCATTTTTTTTGTAATTAATATATTTTCTAATAAATTTATTATTATCAATTTTTTCTTTATTATTATTTTCATTAATATTTAAAATGTATTTGTTTCCATCATAGTTCCTTTTAACTTTTACTTTCCGTTTTATTTTAAAATCTTTTTTATCACGAACTTCATACCATCTCTTATGTCCAAATAAATTTCCCATTCCAATCTCCTCGTTTCTACTTTAATCTAATAAAATATTTTTAAATTAAATCAATTTTACATCTTTCTAATCAAAAATACAATTTTCCATTTTTAGTATACCACATCAATATTAAATCTCAAAAAAATAAGGAGCCGTCAAACATAGCTCCCTACTTCTATTTACTCATAATCCCCATCTATCCTTACTTTTCGTAAAATCAATCCTTCTTTCGCCTTTAGATCCAACTTAATTTTCCCATTTGAACCTGTTCTAAATGTTCTGCCTTCTGTTACCAAATCAATAAATCTTTCATCCTGATAATTTGTTTCAAATTCCACATTTTCCCAGCTGTTAAACGAATTATTTATTACAACAATAATTAAATGATCCTCGATTACTCTTTCATACACAATTATTT

Example 3: Further Evaluation of C2c1p and Associated Components

The Applicants demonstrated in the ensuring experiments that the Class 2CRISPR-Cas loci are expressed to produce mature crRNA and encodeputative tracrRNAs and functional RNA-guided nucleases.

Applicants analyzed a representative C2c1 locus, i.e., theAlicyclobacillus acidoterrestris C2c1 (AacC2c1) CRISPR locus, and haveperformed RNA-sequencing (RNA-seq) and Northern blotting to characterizeits transcriptional activity.

For RNA-sequencing, Alicyclobacillus acidoterrestris (ATCC 49025) wascultured using ATCC Medium 1655 in suspension at 50° C. and 300 rpm. E.coli containing heterologous constructs were cultured in Luria brothsupplemented with appropriate antibiotics in suspension at 37° C. and300 rpm. Both strains were grown in aerobic conditions and harvested instationary growth phase.

To generate the AacC2c1 locus for heterologous expression, genomic DNAwas purified from Alicyclobacillus acidoterrestris using the QiagenDNeasy Blood and Tissue kit and the C2c1 locus was PCR amplified forcloning into pACYC-184.

RNA was isolated from stationary phase bacteria by first resuspendingthe bacteria in TRIzol and then homogenizing the bacteria withzirconia/silica beads (BioSpec Products) in a BeadBeater (BioSpecProducts) for 7 one-minute cycles. Total RNA was purified fromhomogenized samples with the Direct-Zol RNA miniprep protocol (Zymo),DNase treated with TURBO DNase (Life Technologies) and 3′dephosphorylated with T4 Polynucleotide Kinase (New England Biolabs).rRNA was removed with the bacterial Ribo-Zero rRNA removal kit(Illumina). RNA sequencing libraries were prepared from rRNA-depletedRNA using a derivative of the previously described CRISPR RNA sequencingmethod (Heidrich et al., 2015, Methods Mol Biol, vol. 1311, 1-21).Briefly, transcripts were poly-A tailed with E. coli Poly(A) Polymerase(New England Biolabs), ligated with 5′ RNA adapters using T4 RNA Ligase1 (ssRNA Ligase), High Concentration (New England Biolabs), and reversetranscribed with AffinityScript Multiple Temperature ReverseTranscriptase (Agilent Technologies). cDNA was PCR amplified withbarcoded primers using Herculase II polymerase (Agilent Technologies).

The prepared cDNA libraries were sequenced on an MiSeq (Illumina). Readsfrom each sample were identified on the basis of their associatedbarcode and aligned to the appropriate RefSeq reference genome using BWA(Li and Durbin, 2009, Bioinformatics, vol. 25, 1754-1760). Paired-endalignments were used to extract entire transcript sequences using Picardtools (broadinstitute.github.io/picard) and these sequences wereanalyzed using Geneious 8.1.5.

Whole-transcriptome RNAseq of Aac showed transcription of CRISPR arrayis in the same orientation as the Cas genes (FIG. 17). The abundance oftranscripts mapping to the CRISPR array decreased in the 5′ to 3′direction (FIG. 46A). This is in agreement with Applicants' northernresults. The CRISPR array showed robust processing of crRNAs that are 34nt in length with a 5′ 14-nt direct repeat (DR) and a 20-nt spacer (FIG.46A).

The Applicants identified an abundant 79-nt small RNA encoded betweenthe cas2 gene and the CRISPR array and transcribed in the sameorientation as the CRISPR array (FIGS. 46A and B). The internal regionof this small RNA contains a sequence that is highly complementary tothe processed CRISPR repeat sequence (anti-repeat), suggesting that thissmall transcript is the tracrRNA. In silico co-folding of the processed14-nt CRISPR repeat with this putative tracrRNA produces a stablepredicted secondary structure (FIG. 46C).

The Applicants also expressed the Alicyclobacillus acidoterrestris (ATCC49025) C2c1 locus in E. coli and analyzed its expression using Northernblotting. The procedure was performed essentially as described inPougach and Severinov, 2012 (Methods Mol Biol, vol. 905, 73-86). E. coliBL21 A1 cells were transformed with the plasmid pACYCduet-1 containingunder inducible T7 promoter Alicyclobacillus acidoterrestris cas operonand plasmid pCDF-1b containing the minimal CRISPR cassette with a singlespacer. Total RNA was extracted from 5 mL of E. coli cells induced with1 mM arabinose/0.2 mM IPTG and grown until OD₆₀₀, 0.8-1.0. The cellswere lysed by 5-minute treatment using Max Bacterial Enhancement Reagentfollowed by RNA purification with the TRIzol reagent (Thermo FisherScientific). 15 μg of total RNA were separated on a denaturing 8 Murea—12% polyacrylamide gel and electrophoretically transferred toHybond-XL membrane (GE Healthcare) using a Mini Trans-BlotElectrophoretic Transfer Cell (Bio-Rad). The membrane was dried and thenUV cross-linked. ExpHyb hybridization solution (Clontech) was used forhybridization according to manufacturer's instructions for 1 hour at 40°C. with “P-end labeled oligonucleotide probes.

Based on the observation that the putative tracrRNA in A.acidoterrestris contains a characteristic anti-repeat sequence, theApplicants sought to predict potential tracrRNAs for the rest of theidentified C2c1, as well as C2c2 and C2c3, loci by searching foranti-repeat sequences within each locus. In many CRISPR-Cas loci, therepeat located at the promoter-distal end of the CRISPR array isdegenerate and has a sequence that is clearly different from the rest ofrepeats (Biswas et al., 2014, Bioinformatics, vol. 30, 1805-1813). Suchdegenerate repeats were detected in several C2c1 and C2c2 systems (FIGS.9, 14 and 15), allowing the Applicants to predict the direction of thearray transcription. By integrating this information, putative tracrRNAsfor 4 of the 13 C2c1 loci and 4 of the 17 C2c2 loci were identified(FIGS. 9, 14 and 15, FIG. 41A-M-2, FIG. 42A-N-2). In some subtype II-Band II-C loci, the CRISPR array is transcribed in the oppositedirection, starting from the degenerate repeat (Sampson et al., 2013,Nature, vol. 497, 254-257; Zhang et al., 2013, Mol Cell, vol. 50,488-503). Accordingly, we attempted to predict the tracrRNA in differentpositions with respect to the CRISPR array but were unable to identifyadditional candidate tracrRNA sequences. Conceivably, the prediction oftracrRNA for other loci was hampered by a combination of factors such asimperfect complementarity to repeats, lack of an associated CRISPRarray, and/or potential incompleteness of the loci. Furthermore, thepossibility remains that not all Class 2 CRISPR systems requiretracrRNA.

Given the robust expression of transcripts at the Aac locus and theidentification of processed tracrRNA and crRNAs, the Applicants soughtto test the functionality of the AacC2c1 enzyme. To this end, aninterference screen was designed to determine if these loci are activeand to identify the necessary protospacer adjacent motif (PAM), whichdictates where the effector protein will cleave in known Type II systems(FIG. 47A). A library of plasmids conferring ampicillin resistance andcarrying a protospacer flanked by 7 randomized nucleotides wasconstructed. Although the presence of a fragment of DR at the 5′ endside of crRNA spacer suggests a 5′ PAM, separate libraries withrandomized 7-nt sequences positioned upstream or downstream of theprotospacer were tested. More than 107 colonies were pooled to representall 16,384 possible PAM sequences. E. coli cells carrying either theAacC2c1 locus plasmid (FIG. 46D) or a control plasmid (pACYC184) weretransformed with the 5′ or 3′ PAM libraries.

In particular, the randomized PAM plasmid libraries were constructedusing synthesized oligonucleotides (IDT) consisting of 7 randomizednucleotides either upstream or downstream of the spacer 1 target (. Therandomized ssDNA oligos were made double stranded by annealing to ashort primer and using the large Klenow fragment for second strandsynthesis. The dsDNA product was assembled into a linearized PUC19 usingGibson cloning. Stabl3 E. coli cells were transformed with the clonedproducts and more than 10⁷ cells were collected and pooled. Plasmid DNAwas harvested using a Qiagen maxi-prep kit. 360 ng of the pooled librarywas transformed into E. coli cells transformed with the AacC2C1 locus,and pACYC-184. After transformation, cells were plated on LB-agar platessupplemented with ampicillin and chloramphenicol and grown at 37° C.After 16 hours of growth, >4*10⁶ cells were harvested and plasmid DNAwas extracted using a Qiagen maxi-prep kit. The target PAM region wasamplified and sequenced using an Illumina MiSeq with single-end 150cycles.

Under this experimental setup, protospacers with PAMs that arerecognized by the AacC2c1 complex will be destroyed, rendering cellssensitive to ampicillin, and thus are expected to show depletion in thescreen. After overnight growth of the bacteria, the target plasmids wereextracted and deep sequenced. The effectiveness of each PAM wasdetermined by calculating its relative level in the AacC2c1 locus sampleversus the control sample.

Whereas the 3′ PAM screen showed no significant depletion of PAMs, the5′ PAM library screening resulted in the identification of 364 PAMs thatwere significantly depleted; all these PAMs had the sequence NNNNTTN(FIG. 47B). Although there was a slight preference for bases other thanC in the 5′ position immediately adjacent to the protospacer, theseresults indicate that the 5′ TTN motif is recognized by the AacC2c1complex.

The proposed PAM was validated using the first spacer of the AacC2c1locus and all four TTN PAMs (FIG. 47C). Sequences corresponding to bothPAMs and non-PAMs were cloned into digested pUC19 and ligated with T4ligase (Enzymatics). Competent E. coli with either the AacC2c1 locusplasmid or pACYC184 control plasmid were transformed with 20 ng of PAMplasmid and plated on LB agar plates supplemented with ampicillin andchloramphenicol. After 18 hours, colonies were counted with OpenCFU(Geissmann 2013, PLoS ONE, vol. 8, e54072). The results of thisexperiment confirm that a 5′ TTN PAM is necessary for interference andthat interference is slightly reduced with the 5′TTC PAM (FIG. 47C).

The Applicants then sought to investigate whether C2c1 nuclease could beexpressed using human cells and simultaneously explore its tracrRNArequirements. To these ends, the Applicants generated a humancodon-optimized AacC2c1 DNA construct and harvested protein lysate fromHEK293 cells expressing the protein. In particular, C2c1 proteins codonoptimized for human expression and carrying an N-terminal nuclearlocalization tag were designed. The protein sequences were synthesizedand cloned into the pcDNA3.1 expression plasmid by Genscript. UsingLipofectamine 2000 reagent (Life Technologies), 2,000 ng of theexpression plasmids were transfected into HEK293FT cells (6-well platesat 90% confluence). After 48 hours, cells were washed with DPBS (LeftTechnologies), lysed using lysis buffer [20 mM Hepes pH 7.5, 100 mM KCl,5 mM MgCl₂, 1 mM DTT, 5% glycerol, 0.1% Triton X-100, 1× cOmpleteProtease Inhibitor Cocktail Tablets (Roche)]. After sonicating for 10minutes in a Bioruptor sonicators (Diagenode) (50% duty cycle on high),the lysate was centrifuged and the supernatant was frozen at −80° C. forlater use in cleavage assays.

Based on the longest crRNA transcript observed by RNA-sequencing, theApplicants designed a crRNA with a 22-nt direct repeat (DR) and 20-ntspacer targeting a 639-bp region of the EMX1 locus that was PCRamplified from human genomic DNA. Because A. acidoterrestris optimallygrows at 50° C. (Chang and Kang, 2004, Crit Rev Microbiol, vol. 30,55-74), the Applicants hypothesized that the AacC2c1 protein couldperform best at this temperature. Hence, cleavage was performed usingmammalian lysate from cells expressing C2c1 protein at 50° C., unlessotherwise noted, in cleavage buffer (NEBuffer 3, 5 mM DTT) for 1 hour.Each cleavage reaction used 200 ng of target DNA and an equimolar ratioof crRNA:tracrRNA (500 ng of crRNA). The RNA was pre-annealed by heatingto 95° C. and slowly cooling to 4° C. Target DNA consisted of eithergenomic PCR amplicons from the EMX1 gene or the first protospacer of theAacC2c1 locus cloned into pUC19. Reactions were cleaned up using PCRpurification columns (Qiagen) and run on 2% agarose E-gels (LifeTechnologies).

At 50° C., the Applicants showed that the longest expressed tracrRNAobserved in RNAseq experiments (151-nt) is necessary for DNA cleavageusing C2c1 cell lysate and that the cleavage is specific towards theregion of complementarity between the crRNA and the target DNA (FIG.48A). Because RNA-seq experiments yielded putative tracrRNA transcriptsof variable length (FIG. 46A), we tested a series of tracrRNA 3′truncations and found that the shortest tracrRNA allowing for cleavageusing C2c1 cell lysate was a 78-nt species (FIG. 48B). Using thisminimal tracrRNA, we showed that 50° C. is indeed the optimal cleavagetemperature and that there is no observable cleavage below 40° C. usingC2c1 cell lysate (FIG. 48C).

To further validate the PAM requirements of C2c1, the Applicantsdesigned crRNAs towards the protospacer-1 PAM validation constructs usedin the experiments described above (FIG. 47C) and demonstrated cleavagefor the TTT, TTA, and TTC PAMs but not GGA using the 78-nt tracrRNA(FIG. 48D).

Given the detection of the sequence-specific, re-targetable DNA cleavageactivity for the AacC2c1 nuclease, the Applicants hypothesized that,similar to Cas9, the C2c1 crRNA:tracrRNA duplex could be redesigned intoa single-guide RNA (sgRNA) by attaching the 3′ of the 78-nt tracrRNA tothe 5′ end of the crRNA (FIG. 48E). A target cleavage activity similarto that obtained with the crRNA:tracrRNA duplex was observed for thesgRNA with both the EMX1 and protospacer-1 plasmid targets (FIG. 48F).These experiments demonstrated that cell lysate from human cellsexpressing C2c1 can cleave target DNA, identified the temperatureoptimum of the enzyme and demonstrated the requirement for acrRNA:tracrRNA duplex or a single-guide RNA and 5′ PAM for AacC2c1nuclease activity.

In further experiments, Applicants study the domain structure andorganization as well as expression of the CRISPR array in differentbacterial strains comprising CRISPR loci denoted as subtype V-B, e.g asindicated in FIGS. 8A and 8B. Applicants have optimized methods toobtain C2c1 loci from other bacterial strains.

In further experiments, Applicants design pACYC cloning from isolatedgenomic bacterial strains and grown bacterial strains to perform DNA/RNAcutting experiments in E. coli with the effector proteins encoded by theCRISPR loci.

In further experiments, Applicants design PAM bait libraries for DNAcutting to evaluate the cutting ability of the C2c1p effector protein asdescribed herein. Applicants design DNA cutting experiments based on thecutting of a resistance gene transcript.

In further experiments, Applicants test for function in mammalian cellsusing U6 PCR products: spacer (DR-spacer-DR) (in certain aspects spacersmay be referred to as crRNA or guide RNA or an analogous term asdescribed in this application) and tracr for following strains indicatedunder subtype V-B in FIGS. 8A and 8B.

Example 4: Further Evaluation of Ortholog C2c1 Loci

The Applicants also screened the C2c1 locus from Bacillusthermoamylovorans (Bth). Applicants expressed the B. ThermoamylovoransC2c1 (BthC2c1) locus in E. coli and collected RNA-Seq data showingtranscript generation. The BthC2c1 locus was synthesized by Genscriptinto a pET-28 vector. Cells harboring plasmids were made competent usingthe Z-competent kit (Zymo). E. coli containing heterologous constructswere cultured in Luria broth supplemented with appropriate antibioticsin suspension at 37° C. and 300 rpm. The bacteria were grown in aerobicconditions and harvested in stationary growth phase.

RNA was isolated and RNA-sequencing was performed using the methods setforth in Example 3.

Whole-transcriptome sequencing of a synthesized BthC2c1 locus clonedinto pET-28 in E. coli revealed strong processing of both spacerspresent in the array, as well as expression of a 91 nt RNA (FIG. 49A)that displayed secondary structure and repeat-anti-repeat base-paringsimilar to the Aac tracrRNA (FIG. 49B). The putative tracr sequence maybe a −91 bp tracr having the following sequence:

(SEQ ID NO: 79) CGAGGUUCUGUCUUUUGGUCAGGACAACCGUCUAGCUAUAAGUGCUGCAGGGGUGUGAGAAACUCCUAUUGCUGGACGAUGUCUCUUUUAU

In an embodiment of the invention the Direct Repeat sequence (DR) may beprocessed to 15 bp having the sequence UACGAGGCAUUAGCAC (SEQ ID NO: 80).

Applicants determined the consensus PAM for BthC2c1 from the PAMdiscovery screen. The Applicants transformed the PAM library with thecorresponding spacer into E. coli harboring the BthC2c1 locus andcompared depletion to pET-28. The PAM screen was performed as set forthfor the AacC2c1 PAM screen, with the exception that aftertransformation, cells were plated on LB-agar plates supplemented withampicillin and kanamycin. This screen showed that BthC2c1 employs a 5′T-rich PAM with the consensus sequence ATTN (FIG. 49C).

Applicants also expressed the B. sp. C2c1 locus in E. coli and collectedRNA-Seq data showing transcript generation. The arrow in FIG. 50indicates the putative tracrRNA associated with the locus when all thereads are shown. The putative tracr sequence may be a ˜101 bp tracrhaving the following sequence:

(SEQ ID NO: 81) UCAGUCGCACGCUAUAGGCCAUAAGUCGACUUACAUAUCCGUGCGUGUGCAUUAUGGGCCCAUCCACAGGUCUAUUCCCACGGAUAAUCACGACUUUCCA C

In an embodiment of the invention the Direct Repeat sequence (DR) may beprocessed to 20 bp having the sequence GAAAGCUUCGUGGUUAGCAC (SEQ ID NO:82) (FIG. 51). Applicants also showed the co-folding of the DR and thetracrRNA associated with the B. sp. C2c1 locus (FIG. 52).

Example 5: The Adaptation Modules of the Novel Class 2 Systems Appear tohave Evolved Independently from Different Divisions of Class 1

Cas1, a specialized DNAse and integrase that plays a central role inCRISPR adaptation (Nunez et al., 2014, Nature structural & molecularbiology, vol. 21, 528-534; Nunez et al., 2015, , vol. 519, 193-8), isthe most conserved Cas protein (Takeuchi et al., 2012, J Bacteriol, vol.194, 1216-1225) and the only one for which comprehensive phylogeneticanalysis is feasible (Makarova et al. 2011b, Nat Rev Microbiol, vol. 9,467-477; Makarova et al., 2015). In the phylogenetic tree of Cas1,putative subtype V-B (C2c1) is largely monophyletic and clusters withtype I-U (FIGS. 10A and 10B, FIG. 10C-1-W). Among all the (putative)CRISPR-Cas loci, only type I-U and C2c1 encode a fusion of Cas1 and Cas4proteins. Together, this shared derived character and the phylogeneticaffinity of Cas1 strongly suggest that the adaptation module of subtypeV-B derives from that of type I-U. The type V-C Cas1 is the mostdiverged variant of Cas1 sequences discovered to date as indicated bythe long branch in the phylogenetic tree (FIGS. 10A and 10B). In theCas1 tree, the type V-C branch is lodged within subtype I-B (FIGS. 10Aand 10B) although the position of such a fast evolving group should betaken with caution. The type VI Cas1 proteins are distributed among twoclades. The first clade includes Cas1 from Leptotrichia and is locatedwithin the type II subtree along with a small type III-A branch. Thesecond clade includes Cas1 proteins from C2c2 loci of Clostridia andbelongs to a mixed branch that mostly contains Cas1 proteins from typeIII-A CRISPR-Cas systems (FIGS. 10A and 10B).

Although Cas2 is a small and relatively poorly conserved protein, forwhich a reliable phylogeny is difficult to obtain, all available datapoint to the evolutionary coherence of the adaptation module, i.e.,coevolution of the cas1 and cas2 genes (Chylinsky et al., 2014, NucleicAcids Res., vol. 42, 6091-105; Norais et al., 2013, RNA Biol, vol. 10,659-670). Taken together, and without being limited by any hypothesis,these findings indicate that the adaptation modules of the new Class 2CRISPR-Cas systems come from different variants of Class1.

The invention is further described by the following numbered paragraphs:

1. A method of modifying a target locus of interest, the methodcomprising delivering to said locus a non-naturally occurring orengineered composition comprising a Type V CRISPR-Cas loci effectorprotein and one or more nucleic acid components, wherein at least theone or more nucleic acid components is engineered and the effectorprotein forms a complex with the one or more nucleic acid components andupon binding of the said complex to the target locus of interest theeffector protein induces a modification of the target locus of interest,wherein the Type V CRISPR-Cas loci effector protein comprises C2c1p orC2c3p.2. The method of numbered paragraph 1, wherein the target locus ofinterest comprises DNA.3. The method of numbered paragraph 1 or 2, wherein the modification ofthe target locus of interest comprises a strand break.4. The method of numbered paragraph 1, 2 or 3 wherein the effectorprotein is encoded by a subtype V-B CRISPR-Cas loci.5. The method of numbered paragraph 1, 2 or 3 wherein the effectorprotein comprises C2c1p.6. The method of numbered paragraph 1, 2 or 3 wherein the effectorprotein is encoded by a subtype V-C CRISPR-Cas loci.7. The method of numbered paragraph 1, 2 or 3 wherein the effectorprotein comprises C2c3p.8. The method of any of the preceding numbered paragraphs, wherein thetarget locus of interest is comprised in a DNA molecule in vitro.9. The method of any of the preceding numbered paragraphs, wherein thetarget locus of interest is comprised in a DNA within a cell.10. The method of numbered paragraph 9, wherein the cell comprises aprokaryotic cell.11. The method of numbered paragraph 9, wherein the cell comprises aeukaryotic cell.12. The method of any one of the preceding numbered paragraphs, whereinthe target locus of interest comprises a genomic locus of interest.13. The method of any one of the preceding numbered paragraphs, whereinwhen in complex with the effector protein the nucleic acid component(s)is capable of effecting or effects sequence specific binding of thecomplex to a target sequence of the target locus of interest.14. The method of any one of the preceding numbered paragraphs, whereinthe nucleic acid component(s) comprise a putative CRISPR RNA (crRNA)sequence and/or a putative trans-activating crRNA (tracr RNA) sequence.15. The method of any one of numbered paragraphs 1 to 13, wherein thenucleic acid component(s) comprise a putative CRISPR RNA (crRNA)sequence and do not comprise any putative trans-activating crRNA (tracrRNA) sequence.16. The method of any one of numbered paragraphs 2 to 15, wherein thestrand break comprises a single strand break.17. The method of any one of numbered paragraphs 2 to 15, wherein thestrand break comprises a double strand break.18. The method of any one of the preceding numbered paragraphs, whereinthe effector protein and nucleic acid component(s) are provided via oneor more polynucleotide molecules encoding the polypeptides and/or thenucleic acid component(s), and wherein the one or more polynucleotidemolecules are operably configured to express the polypeptides and/or thenucleic acid component(s).19. The method of numbered paragraph 18, wherein the one or morepolynucleotide molecules comprise one or more regulatory elementsoperably configured to express the polypeptides and/or the nucleic acidcomponent(s), optionally wherein the one or more regulatory elementscomprise inducible promotors.20. The method of numbered paragraph 18 or 19, wherein the one or morepolynucleotide molecules are comprised within one or more vectors.21. The method of any one of numbered paragraphs 18 to 20 wherein theone or more polynucleotide molecules are comprised in a delivery system,or the method of numbered paragraph 20 wherein the one or more vectorsare comprised in a delivery system.22. The method of any one of the preceding numbered paragraphs, whereinthe non-naturally occurring or engineered composition is delivered via adelivery vehicle comprising liposome(s), particle(s), exosome(s),microvesicle(s), a gene-gun or one or more viral vectors.23. A non-naturally occurring or engineered composition comprising acomposition having the characteristics of a non-naturally occurring orengineered composition as defined in any one of the preceding numberedparagraphs.24. A non-naturally occurring or engineered composition comprising aType V CRISPR-Cas loci effector protein and one or more nucleic acidcomponents, wherein the effector protein forms a complex with the one ormore nucleic acid components, at least the one or more nucleic acidcomponents is engineered, and upon binding of the said complex to thetarget locus of interest the effector protein induces a modification ofthe target locus of interest, wherein the Type V CRISPR-Cas locieffector protein comprises C2c1p or C2c3p.25. The composition of numbered paragraph 24, wherein the target locusof interest comprises DNA.26. The composition of numbered paragraph 24 or 25, wherein themodification of the target locus of interest comprises a strand break.27. The composition of numbered paragraph 24, 25 or 26 wherein theeffector protein is encoded by a subtype V-B CRISPR-Cas loci.28. The composition of numbered paragraph 24, 25 or 26 wherein theeffector protein comprises C2c1p.29. The composition of numbered paragraph 24, 25 or 26, wherein theeffector protein is encoded by a subtype V-C CRISPR-Cas loci.30. The composition of numbered paragraph 24, 25 or 26, wherein theeffector protein comprises C2c3p.31. The composition of any of numbered paragraphs 24-30, wherein thetarget locus of interest is comprised in a DNA molecule in vitro.32. The composition of any of numbered paragraphs 24-30, wherein thetarget locus of interest is comprised in a DNA within a cell.33. The composition of numbered paragraph 32, wherein the cell comprisesa prokaryotic cell.34. The composition of numbered paragraph 32, wherein the cell comprisesa eukaryotic cell.35. The composition of any one of numbered paragraphs 24-34, wherein thetarget locus of interest comprises a genomic locus of interest.36. The composition of any one of the preceding numbered paragraphs,wherein when in complex with the effector protein the nucleic acidcomponent(s) is capable of effecting or effects sequence specificbinding of the complex to a target sequence of the target locus ofinterest.37. The composition of any one of numbered paragraphs 24-36, wherein thenucleic acid component(s) comprise a putative CRISPR RNA (crRNA)sequence and/or a putative trans-activating crRNA (tracr RNA) sequence.38. The composition of any one of numbered paragraphs 24-36, wherein thenucleic acid component(s) comprise a putative CRISPR RNA (crRNA)sequence and do not comprise any putative trans-activating crRNA (tracrRNA) sequence.39. The composition of any one of numbered paragraphs 25 to 38, whereinthe strand break comprises a single strand break.40. The composition of any one of numbered paragraphs 25 to 38, whereinthe strand break comprises a double strand break.41. The composition of any one of numbered paragraphs 24-40, wherein theeffector protein and nucleic acid component(s) are provided via one ormore polynucleotide molecules encoding the polypeptides and/or thenucleic acid component(s), and wherein the one or more polynucleotidemolecules are operably configured to express the polypeptides and/or thenucleic acid component(s).42. The composition of numbered paragraph 41, wherein the one or morepolynucleotide molecules comprise one or more regulatory elementsoperably configured to express the polypeptides and/or the nucleic acidcomponent(s), optionally wherein the one or more regulatory elementscomprise inducible promotors.43. The composition of numbered paragraph 41 or 42, wherein the one ormore polynucleotide molecules are comprised within one or more vectors.44. The composition of any one of numbered paragraphs 41 to 43 whereinthe one or more polynucleotide molecules are comprised in a deliverysystem, or the composition of numbered paragraph 43 wherein the one ormore vectors are comprised in a delivery system.45. The composition of any one of numbered paragraphs 24-45, wherein thenon-naturally occurring or engineered composition is delivered via adelivery vehicle comprising liposome(s), particle(s), exosome(s),microvesicle(s), a gene-gun or one or more viral vectors.46. A vector system comprising one or more vectors, the one or morevectors comprising one or more polynucleotide molecules encodingcomponents of a non-naturally occurring or engineered composition whichis a composition having the characteristics as defined in any one ofnumbered paragraphs 1 to 45.47. A delivery system comprising one or more vectors or one or morepolynucleotide molecules, the one or more vectors or polynucleotidemolecules comprising one or more polynucleotide molecules encodingcomponents of a non-naturally occurring or engineered composition whichis a composition having the characteristics as defined in any one ofnumbered paragraphs 1 to 45.48. The non-naturally occurring or engineered composition, vectorsystem, or delivery system of any of the preceding numbered paragraphsfor use in a therapeutic method of treatment.49. The non-naturally occurring or engineered composition, vectorsystem, or delivery system of numbered paragraph 48, wherein saidtherapeutic method of treatment comprises gene or genome editing, orgene therapy.50. A eukaryotic cell comprising a modified target locus of interest,wherein the target locus of interest has been modified according to amethod or via use of a composition of any one of the preceding numberedparagraphs.51. The eukaryotic cell according to numbered paragraph 50, wherein themodification of the target locus of interest results in:

-   -   the eukaryotic cell comprising altered expression of at least        one gene product;    -   the eukaryotic cell comprising altered expression of at least        one gene product, wherein the expression of the at least one        gene product is increased;    -   the eukaryotic cell comprising altered expression of at least        one gene product, wherein the expression of the at least one        gene product is decreased; or    -   the eukaryotic cell comprising an edited genome.        52. The eukaryotic cell according to numbered paragraphs 50 or        51, wherein the eukaryotic cell comprises a mammalian cell.        53. The eukaryotic cell according to numbered paragraph 52        wherein the mammalian cell comprises a human cell.        54. The non-naturally occurring or engineered composition,        vector system, or delivery system of any of the preceding        numbered paragraphs, for use in:    -   site-specific gene knockout;    -   site-specific genome editing;    -   DNA sequence-specific interference; or    -   multiplexed genome engineering.        55. A cell line of or comprising the cell according to any one        of numbered paragraphs 50-53, or progeny thereof.        56. A multicellular organism comprising one or more cells        according to any one of numbered paragraphs 50-53.        57. A plant or animal model comprising one or more cells        according to any one of numbered paragraphs 50-53.        58. A gene product from a cell of any one of numbered paragraphs        50-53 or the cell line of numbered paragraph 55 or the organism        of numbered paragraph 56 or the plant or animal model of        numbered paragraph 57.        59. The gene product of numbered paragraph 58, wherein the        amount of gene product expressed is greater than or less than        the amount of gene product from a cell that does not have        altered expression or edited genome.        60. The gene product of numbered paragraph of numbered paragraph        58, wherein the gene product is altered in comparison with the        gene product from a cell that does not have altered expression        or edited genome.        61. A cell modified according to the method, or engineered to        comprise or express the composition or a component thereof of        any one of the preceding numbered paragraphs.        62. An engineered, non-naturally occurring Clustered Regularly        Interspersed Short Palindromic Repeat (CRISPR)-CRISPR associated        (Cas) (CRISPR-Cas) system comprising    -   a) one or more Type V CRISPR-Cas polynucleotide sequences        comprising a guide RNA which comprises a guide sequence linked        to a direct repeat sequence, wherein the guide sequence is        capable of hybridizing with a target sequence, or one or more        nucleotide sequences encoding the one or more Type V CRISPR-Cas        polynucleotide sequences, and    -   b) a C2c1 effector protein or a C2c3 effector protein, or one or        more nucleotide sequences encoding the C2c1 effector protein or        the C2c3 effector protein;    -   wherein the one or more guide sequences hybridize to said target        sequence, said target sequence is 3′ of a Protospacer Adjacent        Motif (PAM), and said guide RNA forms a complex with the C2c1 or        the C2c3 effector protein.        63. An engineered, non-naturally occurring Clustered Regularly        Interspersed Short Palindromic Repeat (CRISPR)-CRISPR associated        (Cas) (CRISPR-Cas) vector system comprising one or more vectors        comprising    -   c) a first regulatory element operably linked to one or more        nucleotide sequences encoding one or more Type V CRISPR-Cas        polynucleotide sequences comprising a guide RNA which comprises        a guide sequence linked to a direct repeat sequence, wherein the        guide sequence is capable of hybridizing with a target sequence,    -   d) a second regulatory element operably linked to a nucleotide        sequence encoding a C2c1 or C2c3 effector protein;    -   wherein components (a) and (b) are located on the same or        different vectors of the system,    -   wherein when transcribed, the one or more guide sequences        hybridize to said target sequence, said target sequence is 3′ of        a Protospacer Adjacent Motif (PAM), and said guide RNA forms a        complex with the C2c1 or C2c3 effector protein.        64. The system of numbered paragraph 62 or 63 wherein the target        sequences is within a cell.        65. The system of numbered paragraph 62 or 63 wherein the cell        comprises a eukaryotic cell.        66. The system according to numbered paragraph 62 or 63, wherein        when transcribed the one or more guide sequences hybridize to        the target sequence and the guide RNA forms a complex with the        C2c1 or C2c3 effector protein which causes cleavage distally of        the target sequence.        67. The system according to numbered paragraph 66, wherein said        cleavage generates a staggered double stranded break with a 4 or        5-nt 5′ overhang.        68. The system according to numbered paragraph 62 or 63, wherein        the PAM comprises a 5′ T-rich motif.        69. The system according to numbered paragraph 62 or 63, wherein        the effector protein is a C2c1 effector protein derived from a        bacterial species selected from the group consisting of        Alicyclobacillus acidoterrestris (e.g., ATCC 49025),        Alicyclobacillus contaminans (e.g., DSM 17975), Desulfovibrio        inopinatus (e.g., DSM 10711), Desulfonatronum thiodismutans        (e.g., strain MLF-1), Opitutaceae bacterium TAV5, Tuberibacillus        calidus (e.g., DSM 17572), Bacillus thermoamylovorans (e.g.,        strain B4166), Brevibacillus sp. CF112, Bacillus sp. NSP2.1,        Desulfatirhabdium butyrativorans (e.g., DSM 18734),        Alicyclobacillus herbarius (e.g., DSM 13609), Citrobacter        freundii (e.g., ATCC 8090), Brevibacillus agri (e.g., BAB-2500),        Methylobacterium nodulans (e.g., ORS 2060).        70. The system according to numbered paragraph 69, wherein the        PAM sequence is TTN, where N is A/C/G or T and the effector        protein is AacC2c1 or wherein the PAM sequence is TTTV, where V        is A/C or G and the effector protein is AacC2 c1.        71. The system according to numbered paragraph 62 or 63, wherein        the C2c1 or C2c3 effector protein comprises one or more nuclear        localization signals.        72. The system according to numbered paragraph 62 or 63, wherein        the nucleic acid sequences encoding the C2c1 effector protein or        the C2c3 effector protein is codon optimized for expression in a        eukaryotic cell.        73. The system according to numbered paragraph 62 or 63 wherein        components (a) and (b) or the nucleotide sequences are on one        vector.        74. A method of producing a plant, having a modified trait of        interest encoded by a gene of interest, said method comprising        contacting a plant cell with a system according to numbered        paragraph 62 or 63 or subjecting the plant cell to a method        according to numbered paragraph 1, thereby either modifying or        introducing said gene of interest, and regenerating a plant from        said plant cell.        75. A method of identifying a trait of interest in a plant, said        trait of interest encoded by a gene of interest, said method        comprising contacting a plant cell with a system according to        numbered paragraph 62 or 63 or subjecting the plant cell to a        method according to numbered paragraph 1, thereby identifying        said gene of interest.        76. The method of numbered paragraph 75, further comprising        introducing the identified gene of interest into a plant cell or        plant cell line or plant germplasm and generating a plant        therefrom, whereby the plant contains the gene of interest.        77. The method of numbered paragraph 76, wherein the plant        exhibits the trait of interest.        78. A particle comprising a system according to numbered        paragraph 62 or 63.        79. The particle of numbered paragraph 78, wherein the particle        contains the C2c1 or C2c3 effector protein complexed with the        guide RNA.        80. The system or method of numbered paragraph 1, 62 or 63,        wherein the complex, guide RNA or protein is conjugated to at        least one sugar moiety, optionally N-acetyl galactosamine        (GalNAc), in particular triantennary GalNAc.        81. An engineered, non-naturally occurring composition        comprising a CRISPR-Cas system, said system comprising a        functional Type-V CRISPR-Cas loci effector protein and guide RNA        (gRNA);

wherein the gRNA comprises a dead guide sequence;

whereby the gRNA is capable of hybridizing to a target sequence;

whereby the CRISPR-Cas system is directed to the target sequence withreduced indel activity resultant from nuclease activity of a non-mutantType-V CRISPR-Cas loci effector protein of the system; and

whereby the functional Type-V CRISPR-Cas loci effector protein is C2c1por C2c3p.

82. A method of inhibiting cell growth, the method comprising deliveringto the cell a non-naturally occurring or engineered compositioncomprising a functional Type-V CRISPR-Cas loci effector protein andguide RNA (gRNA);

whereby the gRNA is capable of hybridizing to a target DNA sequence ofthe cell;

whereby the CRISPR-Cas system is directed to the target DNA sequencewith reduced indel activity resultant from nuclease activity of anon-mutant Type-V CRISPR-Cas loci effector protein of the system; and

whereby the functional Type-V CRISPR-Cas loci effector protein is C2c1por C2c3p.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following numbered paragraphs define the scope of the invention andthat methods and structures within the scope of these numberedparagraphs and their equivalents be covered thereby.

What is claimed is:
 1. A Type V non-naturally occurring, engineeredcomposition comprising a) a Type V Cas polypeptide comprising one ormore catalytic motifs of a RuvC nuclease domain but not comprising a HNHdomain, and one or more nucleic acid components comprising aheterologous guide sequence and a tracrRNA sequence that are capable offorming a complex with the Type V Cas polypeptide and directingsite-specific binding of said complex to a target sequence; or b) a TypeV Cas polypeptide comprising one or more catalytic motifs of the RuvCnuclease domain and a Zn finger region with at least one Zn-bindingcysteine, but not comprising a HNH domain, and one or more nucleic acidcomponents comprising a heterologous guide sequence that is capable offorming a complex with the Type V Cas polypeptide and directingsequence-specific binding of said complex to a target sequence.
 2. Thecomposition of claim 1, wherein the Type V Cas polypeptide comprises anarginine rich cluster, but which does not bind Zinc.
 3. The compositionof claim 1, wherein the Type V Cas polypeptide is a C2c1 or C2c3polypeptide.
 4. The composition of claim 3, wherein the C2c1 polypeptideis obtained from a bacterial species selected from the group consistingof Alicyclobacillus acidoterrestris, Alicyclobacillus contaminans,Desulfovibrio inopinatus, Desulfonatronum thiodismutans, Opitutaceaebacterium TAV5, Tuberibacillus calidus, Bacillus thermoamylovorans,Brevibacillus sp. CF112, Bacillus sp. NSP2.1, Desulfatirhabdiumbutyrativorans, Alicyclobacillus herbarius, Citrobacter freundii,Brevibacillus agri, and Methylobacterium nodulans.
 5. The composition ofclaim 3, wherein the C2c1 polypeptide comprises at least 95% sequenceidentity to a C2c1 polypeptide selected from the group consisting of:SEQ ID NOs: 548-567.
 6. The composition of claim 3, wherein the Type VCas polypeptide of a) is C2c1 polypeptide, or the Type V Cas polypeptideof b) is C2c3 polypeptide.
 7. The composition according to claim 1,wherein the Type V Cas polypeptide comprises one or more nuclearlocalization signals.
 8. The composition according to claim 1, whereinthe Type V Cas polypeptide comprises a mutation of one or more aminoacid residues in a catalytically active domain of the effector proteinand has reduced or abolished nuclease activity compared with a Type VCas polypeptide lacking said one or more mutations.
 9. The compositionaccording to claim 1, wherein the Type V Cas polypeptide is linked to aheterologous functional domain.
 10. A vector system comprising one ormore vectors, the one or more vectors comprising one or morepolynucleotide molecules encoding components of the non-naturallyoccurring or engineered composition of claim
 1. 11. The vector systemaccording to claim 10, wherein the one or more polynucleotide moleculescomprise one or more regulatory elements capable of expressing thepolypeptide(s) and/or the nucleic acid component(s), optionally whereinthe one or more regulatory elements comprise inducible promotors. 12.The vector system according to claim 10, wherein the Type V Caspolypeptide is codon optimized for expression in a eukaryotic cell. 13.The method of claim 12, wherein modifying comprises cleaving thesequence.
 14. The method of claim 12, wherein the target sequencecomprises DNA.
 15. An isolated eukaryotic or prokaryotic celltransiently or non-transiently transfected with the vector of claim 10.16. An in vitro, in vivo, or ex vivo method of targeting a targetsequence in a eukaryotic or prokaryotic cell, the method comprising:contacting a sample with the engineered composition of claim
 1. 17. Themethod of claim 16, wherein the sample comprises a polynucleotidecomprising the target sequence in a eukaryotic cell.
 18. The method ofclaim 16, wherein the Type V Cas polypeptide and nucleic acidcomponent(s) are provided via one or more polynucleotide moleculesencoding the polypeptide(s) and/or the nucleic acid component(s), andwherein the one or more polynucleotide molecules are capable ofexpressing the polypeptide(s) and/or the nucleic acid component(s). 19.The method of claim 16, wherein the engineered composition is deliveredvia liposomes, nanoparticles, exosomes, microvesicles, a gene-gun or oneor more viral vectors.
 20. The method of claim 16, further comprisingmodifying said target sequence.